Introduction To Cell & Molecular Biology Techniques: Immunofluorescence

Introduction to Cell & Molecular Biology
Techniques :
Immunofluorescence
DAPI (DNA) GFP γ Tubulin
Merge
© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

SPARQ-ed – University of Queensland Diamantina Institute ph. +61 7 3443 6920 fax. +61 7 3443 6966
University of Queensland, Australia. Email: sparqed@uq.edu.au
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SPARQed is a collaboration between The University of Queensland’s Diamantina Institute and The Queensland
Government’s Department of Education, Training and Employment.
The Immunofluorescence Workshop is based on technique used in the SPARQ-ed Research Immersion
Programs developed by scientists at the University of Queensland Diamantina Institute. The experimental
protocols and all supporting materials were adapted for student use by Dr Peter Darben, under the supervision
of Dr Sandrine Roy.
Cover Image : A field of HeLa cells stained with DAPI (which binds to DNA and fluoresces blue) and treated with
an antibody against γ Tubulin and a secondary antibody conjugated to a red flurophore. Some of these cells
have been transfected with the gene for the jellyfish protein green fluorescent protein (GFP). The figure shows
the three colour channels, imaged separately for blue, green and red, and the combined image obtained when
these three channels are merged. Image courtesy Rose Boutros.
All materials in the manual are Copyright 2014, State of Queensland (Department of Education and Training).
Permission is granted for use in schools and other educational contexts. Permission for use and reproduction
should be requested by contacting Peter Darben at p.darben@uq.edu.au. These materials may not be used for
commercial purposes
Risk assessments were developed with the assistance of Paul Kristensen, Maria Somodevilla-Torres and Jane
Easson.
Many thanks to all in the “Gab Lab” whose patience and support made this happen.

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Table of Contents
Introduction
What is Cell Biology ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Common Cell Biology Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
The Workshop
Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Theoretical Basis of the Project
Tissue Culture and Cancer Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Preparation of Cells for Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Antibodies and Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Experimental Protocols
How to Use this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Preparation of Culture Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Blocking and Permeablisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Treatment with Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Imaging Using Epifluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
What to Look Out For . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Appendices
Appendix A : DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Appendix B : Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Appendix C : Using a Micropipette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Appendix D : Glossary of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

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Introduction
What is Cell Biology ?
Cells form the basis of all living things. They are the smallest single unit of life, from the simplest bacteria to
blue whales and giant redwood trees. Differences in the structure of cells and the way that they carry out their
internal mechanisms form the basis of the first major divisions of life, into the three kingdoms of Archaea
(“ancient” bacteria), Eubacteria (“modern” bacteria) and Eukaryota (everything else, including us). An
understanding of cells is therefore vital in any understanding of life itself.
Cell biology is the study of cells and how they function, from the subcellular processes which keep them
functioning, to the way that cells interact with other cells. Whilst molecular biology concentrates largely on the
molecules of life (largely the nucleic acids and proteins), cell biology concerns itself with how these molecules
are used by the cell to survive, reproduce and carry out normal cell functions.
In biomedical research, cell biology is used to find out more about how cells normally work, and how
disturbances in this normal function can result in disease. An understanding of these processes can lead to
therapies which work by targeting the abnormal function.
Common Cell Biology Techniques
The following list covers some of the more commonly used cell biology techniques – it is by no means
exhaustive.
Cell / Tissue Culture – in the same way that bacteria and other simple organisms can be grown in the
laboratory outside their normal environment, cells and tissues from more complicated organisms can
be cultured as well. The techniques are slightly different, and the culture media are more complex to
reflect the complex internal environment inside the host from which the cells are derived, however
cell and tissue culture is a powerful tool which provides an almost limitless supply of test material for
researchers to use without resorting to using whole organisms. In addition, the controlled conditions
in cell and tissue culture allow researchers to carry out experiments with a lower number of variables
which may affect the outcome of the test. Cell culture may use cells removed directly from an
organism (primary culture), or it may use lines of cultured cancer cells. The benefit of the latter
approach is that cancer cells continue to divide, while primary cultures cease dividing after a number
of cycles.
Microscopy – the basic tool of cell biology is microscopy. Recent advances in imaging technology have
allowed an unprecedented amount of information to be gleaned from microscopic analysis. Types of
microscopic techniques which are used include :
Brightfield – traditional microscopy, where cells are illuminated by visible light. Brightfield
microscopy gives a general picture of cell function, although that information is not very
detailed or specific. As animal cells lack cell walls, brightfield microscopy may use special
techniques such as phase contrast to show cellular structures in more detail. Brightfield
microscopy allows imaging of live or fixed (dead) cells and tissues)

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Electron Microscopy – uses a focused beam of electrons instead of light. Electron microscopy
permits a much higher magnification of specimens than light microscopy and is useful in
obtaining detailed information about sub-cellular structures. Electron microscopy requires
extensive processing and so can only be performed on fixed specimens. Transmission electron
microscopy provides a cross section of a specimen, while scanning electron microscopy gives a
three-dimensional image of the surface of a specimen.
Fluorescence Microscopy – uses fluorescent materials to indicate structures in a specimen.

Fluorescence occurs when light of one wavelength “excites” a material and causes it to emit
light of a different wavelength. Most fluorescent materials give off visible light after excitation
by ultraviolet light. Structures may be naturally fluorescent (autofluorescence) or they may be
labeled with a compound which is fluorescent (eg. DAPI is a dye which binds onto DNA. The
DNA and nuclei of cells stained with DAPI emit a blue light under ultraviolet light).
Immunofluorescence – antibodies are proteins made by the immune system which bind onto
specific parts of proteins. Antibodies can be raised against any protein in the cell. If these
antibodies are attached to a fluorescent tag, the tag will only show up where that antibody
attached (ie. where the target protein is found in the cell). Immunofluorescence allows very
specific targeting of cellular structures.
RNA Interference – RNA interference uses short sequences of RNA which are complementary to the
mRNA which carries to instructions to translate proteins from the DNA to the ribosomes. The
interfering RNA binds to the target sequence, preventing it from being translated. As a result, careful
selection of interfering RNA can be used to silence a particular gene. This allows researchers to study
what role a protein plays in a cell, by observing what happens when that protein is absent.
Timelapse Microscopy – many cellular processes (eg. mitosis) occur over a period of time which is not
practical for direct observation. Imaging cells over a period of time (eg. a photograph is taken every 20
minutes for 24 hours) allows us to combine these images in a “movie” which compresses a long time
period into a shorter one.
The Workshop
Whilst the nucleus and some other organelles are easily visualized using brightfield microscopy, most other
sub-cellular structures cannot be differentiated or even seen using conventional microscopes. Therefore these
structures and biomolecules must be demonstrated using more specific techniques such as
immunofluorescence and fluorescence microscopy.
In this project you will be provided with a coverslip containing cancer cells grown in culture. You will then
expose the cells to antibodies directed against proteins found in subcellular structures, allowing these primary
antibodies to bind to those structures. You will then treat the cells with secondary antibodies directed against
the primary antibodies. These secondary antibodies have been conjugated to a fluorescent dye, effectively
attaching a fluorescent label to the structure you are interested in seeing. You will then use a fluorescence
microscope to image the cells, taking images of each colour channel and then combining the images to create
a merged image which demonstrates all structures.

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Getting Started
Before you begin, make sure that you are familiar with the relevant theory behind the techniques we will be
performing. This manual contains several appendices which will provide you with this information. Make sure
you read this information before proceeding.
Appendix A : DNA
Appendix B : Proteins
Appendix A : Using a Micropipette
Appendix B : Glossary of Terms
Information on other cell and molecular biology concepts and techniques are provided at the SPARQ-ed
website at : http://www.di.uq.edu.au/sparqedservices
Theoretical Basis of the Project
Tissue Culture and Cancer Cell Lines
When researchers want to obtain sufficient quantities of organisms to use in their studies, they may grow
them up in a vat of broth containing all of the nutrients they need to survive. This is called culturing the
organisms, and for many simple, free-living organisms such as bacteria or fungi, the process is relatively
straightforward. These organisms often require very little in the way of specialised nutrients and so long as the
culture conditions are suitable, they will undergo almost constant division, increasing their populations at an
exponential rate.
If cells from a multi-cellular organism like a human are to be used in research, the culture methods are not
nearly so simple. Firstly, cells from multi-cellular organisms have differentiated to such an extent that they can
no longer survive without the complex systems of nutrients and stimuli provided by the other cells in the body.
As a result, growing human cells in culture requires the use of growth media containing a complex mixture of
basic nutrients and specific growth factors provided by the inclusion of serum (the liquid component of clotted
blood). In addition, body cells spend most of their time carrying out the functions which allow them to play
their roles within the body. This means that they are not constantly dividing - in many cases they must be
stimulated to enter mitosis, and in some cases do not divide at all. As a result, human cells in culture often
proliferate very slowly.
One solution to this problem is the use of cancer cells. One of the hallmarks of cancer is the loss of regulation
of the cell cycle, leading to cells constantly passing through division cycles. This results in hyperproliferation of
the cells, which in the body leads to the growth of tissue masses known as tumours. Cancer cell
hyperproliferation means that they can be grown in culture for many generations, with further cultures able to
be sub-cultured from the original – they are effectively immortal. This has led to the production of cancer cell
lines, each of which was derived from a tumour recovered from an individual with cancer.
Most types of cancer have numerous cell lines which researchers can use to study the cancer in question. Each
of these cell lines have particular characteristics dependent on the cancer from which they were derived,
allowing researchers to select the line which most closely matches the situation they are working on.
Unfortunately, being cancer cells, the cells contain errors and abnormalities which mean that they do not
often behave as normal cells do, making them inappropriate models for normal cells, or even for cancers of

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other parts of the body. In addition, the same errors which remove the cell cycle controls in cancer cells may
also remove mechanisms which regulate damage to the DNA, allowing mutations to accumulate in the cells
which make them even more distinct from the cells from which they were derived
HeLa cells are possibly the best known of all cancer cell
Henrietta Lacks
lines. They were originally recovered from a cervical cancer
removed from a patient named Henrietta Lacks in 1951. The patient from
The cancer resulted from an infection by a strain of human whom the HeLa
papillomavirus (HPV18), and so the genome of these cells cell line was derived
also contains the genome for this strain of HPV. HeLa was was Henrietta Lacks,
the first cell line to be successfully and continuously who was living in
cultured in vitro and have been used widely around the
Baltimore in the US
world since the physician who first subcultured them made
them and the techniques used to grow them freely at the time of her
available to scientists around the world. HeLa cells have diagnosis. While the
played an important role in many important medical contribution that the HeLa cell line made
discoveries, including the development of the Polio is well known, it is an interesting case of
vaccine. medical ethics, as permission for the use
of her tissue was never sought. More
In this workshop, you will be using a genetically modified
information about the life of Ms Lacks
strain of HeLa cells used by the Cell Cycle Research Group
at the University of Queensland Diamantina Institute. and the issues around the use of her cells
These cells have a modified gene for one of the histone can be found in the book The Immortal
proteins in the nucleus (H2), where the gene includes a Life of Henrietta Lacks by Rebecca Skloot.
gene derived from a jellyfish (the gene for green
fluorescent protein, or GFP). The histones are a family of proteins around which DNA wraps and which assist in
the packaging of DNA and help to regulate the expression of genes. When the modified gene is expressed, the
protein produced includes a GFP “tag” on the end which glows green when exposed to blue light. This has
produced a cell line in which the nucleus fluoresces green, which means that this green signal can be used in
place of a DNA dye such as DAPI. This fluorescence is produced in living cells, allowing them to be used to
study the function and appearance of the nucleus at different stages of the cell cycle through live cell imaging.
Preparation of Cells for Immunofluorescence
Using routine brightfield microscopy, a reasonable amount of detail in the cell can be determined. Large
structures such as the nucleus (and its nucleoli) and vacuoles are easily distinguished inside the cell with even
fairly rudimentary microscopes. Some other structures can also be demonstrated with the careful use of dyes
and stains which give a colour based on chemical reactions with cellular components, and this forms the basis
of cytochemistry (on individual cells) and histochemistry (on thin sections of tissue). However, even these
methods are not nearly specific enough to properly demonstrate the wide variety of subcellular structures and
materials. Immunofluorescence is a technique which uses the highly specific binding of antibodies to their
target antigens as a way of demonstrating materials and structures inside cells.
Before any immunofluorescence procedures can be carried out on the cells, they must be properly prepared.
For this workshop, the cells have been grown on a coverslip placed on the bottom of a culture plate. When a
new culture is needed, a suspension of cells is prepared with the cells floating around inside the cell culture
medium. The number of cells per unit volume (generally per millilitre of medium) can be calculated using cell
counters and a specific number of cells added to a dish containing culture medium by adding the correct
volume of cell suspension (eg. if the density of cells in the suspension is 100,000 cells / mL and you needed

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100,000 cells to start a culture, you would add 1mL of suspension). The exact seeding density depends on the
reproductive rate of the cells you are using and how dense you want the culture to be in the time you have.
When the cells are added to the culture dish, they sink to the bottom and attach through proteins embedded
in the cell membrane. Cells that have attached to the bottom of the dish take on the appearance of a fried egg,
with the nucleus as the yolk and the cytoplasm as the white. Cells only release from the bottom of the flask
when they are in the process of dividing, becoming spherical as they carry out mitosis. For this workshop, the
cells have been added to a 6-well culture plate. In each well, a number of coverslips were added to the wells
prior to the addition of the cells. When the cells settled to the bottom of the well, some landed on the
coverslip. This means that the cells can be easily removed from the well and treated while on the coverslip,
and eventually the coverslip containing the cells can be mounted onto a microscope slide for observation. The
coverslips have been previously treated with Poly-L-Lysine, a peptide which acts as a cellular “glue” and assists
in holding the cells (including mitotic cells which have rounded up) onto the coverslip.
At several times during the course of the immunofluorescence procedures, cells will need to be washed. This is
usually done by replacing the liquid in the well they are contained in with phosphate buffered isotonic saline, a
solution of sodium chloride at 9g/L in a buffer which keeps the pH at a physiological level of around 7.2.
Because the cells are stuck to the coverslip, the liquid can be gently removed and replaced with a solution
which does not put the cells under osmotic stress.
Once cells die (which starts to occur once their nutrients and ideal growing conditions have been removed),
they start to break down and lose their structure and integrity. A typical live cell taken through the processes
needed for immunofluorescence would have started the breakdown process by the time the techniques have
been completed. Therefore the cells must be preserved prior to undergoing antibody treatment. This process
is called fixation, and is the same technique used to preserve biological specimens in museums. Fixation relies
on “freezing” the proteins in place using a chemical process rather than temperature. The tertiary structure of
proteins is maintained by weak hydrogen bonds between the side chains of the amino acids that make up the
proteins. These hydrogen bonds are easily broken by increases in temperature or changes in pH, resulting in a
loss of protein structure called denaturation. Formalin is a buffered solution of the gas formaldehyde which
forms permanent covalent crosslinks between protein chains and locks them into their tertiary structure,
regardless of minor changes in temperature or pH, and thus preserves the structure of the cell. In this
investigation, you will use a type of formalin called paraformaldehyde to fix the cells, although other fixatives
like ice cold methanol can also be used.
Once cells have been washed, fixed in paraformaldehyde and washed again, they must then be permeablised.
The antibodies used for immunofluorescence are large protein molecules which cannot cross the cell
membrane, so the cell membrane must be removed. It is very important that the cells must be fixed prior to
permeablisation, otherwise the cell will burst.
To understand why it is important to fix the cells, it may help to imagine the cell as a dome tent filled with
cooked spaghetti (with the nucleus as a beachball inside it, perhaps). The shape of the cell is maintained by
structural proteins such as Tubulin, represented in our analogy by the cooked spaghetti, while the whole shape
of the cell is maintained by the cell membrane, represented by the fabric of the tent itself. If you were to cut
away the tent fabric (removing the cell membrane through permeablisation), all of the spaghetti would fall out
and spill onto the ground. Washing the cell would then have the effect of hosing the mess away.
Fixing the cells would be like drying the spaghetti out. It would shrink slightly and revert back to its state prior
to cooking, however it would be in the same position it was in when the drying occurred. The tent fabric (cell
membrane) could then be removed and you would be left with a mound of dried spaghetti in the same shape
as the tent, but without the tent around it (see Figure 1). Chemical fixation does make some changes to the
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proteins in the cell, however enough of the original structure is often left to retain some of the proteins’
functions, including the ability of the proteins to bind antibodies and the fluorescence characteristics of green
fluorescent protein (both of which are vital for this investigation.
Unfixed Cell
Permeablisation
Fixed Cell Permeablisation
Figure 1 - Fate of unfixed and fixed cells following permeablisation
In this workshop, cells will be permeablised using a detergent (Triton X100). Detergents work by solublising
lipids in water, and since the cell membrane is made up of lipid molecules, the Triton X100 in this method
removes the lipids from the membrane, allowing large molecules such as antibodies to enter the cell and bind
to proteins inside. At the same time as they are permeablised, the cells will be blocked. Blocking involves the
addition of a solution of proteins (usually bovine serum albumin, or BSA). Cells may contain proteins which can
bind onto antibodies and other proteins non-specifically, rather than the antibodies specifically binding onto
their target proteins. The BSA “soaks up” these
non-specific binding sites, ensuring that the only Variable Regions
way the antibodies can bind is via their specific
targets. Once permeablised, blocked and washed
once more, the cells are ready to be treated with
antibodies.
Antibodies and Immunofluorescence
Antibodies are large, complex proteins made by Light Chain

specific immune cells in the body (the plasma cells,
which are derived from populations of B
lymphocytes). Antibodies are produced in
response to the presence of an agent in the body Heavy Chain
which the immune system recognizes as being not
from the body. These agents are usually foreign
materials, such as proteins on the surface of
microbes, although in some cases the immune
system may target the body’s own proteins, Figure 2 Generalised Antibody Structure
causing autoimmune diseases such as Type I
Diabetes and Rheumatoid Arthritis.
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Antibodies are composed of four peptide chains (two light chains and two heavy chains) arranged like a capital
letter “Y” (see Figure 2). Most of an antibody’s structure is constant between antibodies (with some variation
between different classes), however at the ends of the “arms” of the Y is a small, highly variable region made
up of a region of the light and heavy chains. It is changes in the shape of this region which determines the
difference between antibodies. The shape of this variable region is such that it fits around and binds very
specifically to a region on the material it is raised against. The area which it binds to is called the epitope, while
the material where the epitope is found is called the antigen. A different antibody is made in response to each
epitope on each antigen, with the variable regions confirming exactly to each epitope. This means that
antibodies are made which bind very specifically to particular epitopes on antigens. This specificity is so high
that two antibodies can be generated which can tell the difference between two versions of the same protein,
one which has a phosphate group attached and one which does not. This high level of specificity makes
antibodies extremely useful in detecting particular proteins, and is used in techniques such as diagnosis of
disease agents in the blood, western blotting, immunohistochemistry, and, of course, immunofluorescence.
Antibodies are made in animals, or in cell lines derived from those animals. To generate an antibody against a
particular protein, samples of that protein are injected into a test animal. A better antibody response is
generated if the target protein comes from a different species to the animal used to raise the antibody as the
host animal would recognize the protein as being foreign. For example, if you are interested in using
antibodies to detect a human protein, you would use a non-human animal such as a mouse, rabbit or goat to
raise the antibody. Once the animal starts making the antibody, it can be recovered from the animal’s blood
and purified for use. In some cases the lines of plasma cells which are making the antibody can be isolated and
fused with a cancer cell, making a hybridoma cell line which has the immortality of the cancer cells and the
antibody-generating properties of the plasma cells. This means that the antibodies can be produced in culture
without the need for using further animals.
The technique you will be using is indirect immunofluorescence. Direct immunofluorescence involves
chemically combining a fluorescent dye directly to the primary antibody raised against the protein you are
interested in. The antibody, with its fluorescent label binds, onto the target protein in the cell, essentially
colouring the target protein with the label (see Figure 3). Direct immunofluorescence is not often used now, as
it would involve making a range of differently labeled antibodies for every protein studied in laboratories. In
addition, the sensitivity of the technique is low, as there is only one label for each binding site.
With indirect immunofluorescence, the primary antibody is left unlabelled. It still binds to the target protein,
however it must be itself demonstrated using a labeled secondary antibody. This secondary antibody is
generated in the same way as the primary antibody, however its target is antibodies from the species in which
the primary antibody has been raised. For example, if the primary antibody was raised in a mouse, the labeled
secondary antibody would be raised in another species against mouse antibodies (eg. goat anti-mouse). This
allows laboratories to have a wide selection of primary antibodies directed against any protein they might be
working on, and then a smaller selection of labeled secondary antibodies directed against the species the
primary antibodies were raised in (eg. anti-mouse, anti-rat, anti-goat, anti-chicken, etc). In addition, because
multiple labeled secondary antibodies can bind to a single primary antibody, the signal strength is higher,
resulting in greater sensitivity (see Figure 4).
If primary antibodies from different species are used, then multiple proteins can be targeted in the one cell.
The only limits are the number of species available and the range of dyes used. For example, a mouse derived
primary antibody against protein A could be used alongside a goat derived primary antibody against protein B,
so long as the anti-mouse and anti-goat secondary antibodies had different coloured labels.

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Antigen Present Antigen Absent
Treatment with labeled antibody
UV Light Unbound antibody washed away UV Light
Fluorescence observed where antigen is located
Figure 3 - Principal of Direct Immunofluorescence

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Treatment with unlabelled primary antibody
Unbound antibody washed away
Treatment with unlabelled secondary antibody
(continued over)
Figure 4 - Principle of Indirect Immunofluorescence

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Unbound secondary antibody washed away

UV Light UV Light
Fluorescence observed wherever antigen is located
Figure 4 - Principle of Indirect Immunofluorescence (Continued)
The general protocol for antibody treatments is to expose the cells to the primary antibody for 1-2 hours, wash
thoroughly in PBS to remove all unbound primary antibody, then expose to the secondary antibody for 1 hour.
The cells are washed in PBS to removed any unbound secondary antibody, and washed in water to remove the
PBS (which forms beautiful, but annoying crystals when the coverslips are mounted). To make a permanent
preparation, the coverslips are mounted on a microscope slide in a special mounting medium which hardens
upon contact with air.
Targets
In this workshop, you will be demonstrating three components of the cells :
 The Nucleus – the HeLa H2B GFP cell line expresses a green fluorescent protein tag on the H2 Histone
protein located in the nucleus. This protein is found throughout the nucleus and remains associated with
the DNA, even during mitosis. These cells therefore show a green fluorescence in the nucleus, and in the
chromosomes during mitosis.

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 α-Tubulin (Alpha Tubulin) – α-Tubulin is one of the subunits of the tubulin protein which forms an
important part of the cytoskeleton which provides support to the cell. During interphase, tubulin is
found throughout the cytoplasm of the cell right up to the cell membrane and has a fibrous appearance.
During mitosis, the cell assembles the mitotic spindle from Tubulin, and this structure appears as a
“birdcage”-like structure on which the chromosomes are arranged. Tubulin is also a major component of
the centrioles which make up the centrosome, and this appears as paired pinpoint bodies within the
cytoplasm of the cell. In this investigation, the antibody against α-Tubulin used will be derived from a
rabbit, and a secondary anti-rabbit antibody conjugated to a far-red (647nm) secondary antibody will be
used to demonstrate it. The human eye cannot detect light at 647nm easily, so a false colour (eg.
magenta) will be applied, and the α-Tubulin will appear as fine fibres of this colour throughout the
cytoplasm. Mitotic spindles will also be demonstrated using this method.
 Complex IV (or Cytochrome C Oxidase) – Complex IV is a large transmembrane protein composed of 13

subunits and found in the mitochondria of eukaryotic cells. Complex IV catalyses the last step in the
electron transport chain in cellular respiration, transferring electrons to molecular oxygen and allowing
the production of water molecules. In eukaryotic cells, apart from a couple of exceptions, Complex IV is
found only in the membranes of mitochondria, which means that this protein complex can be used as a
mitochondrial “marker” – a target which demonstrates the presence of mitochondria in the cell. The
antibody against Complex IV used in this workshop is derived from a mouse, and a secondary anti-
mouse antibody conjugated to a blue (405nm) fluorescent label will be used to demonstrate it. The
mitochondria should appear as blue specks throughout the cytoplasm of the cell.
Fluorescence Microscopy
In routine light microscopy, visible light is passed through a specimen on a microscope slide. This light
continues up through the optics of the microscope to the eyes. Structures in a specimen will interfere slightly
with the passage of the light, absorbing some wavelengths to give it a colour, or subtly bending it to reveal the
shape of the structure. The structures visible through light microscopy may be enhanced through the use of
coloured dyes which bind to components in the specimen, which is the basis of histochemistry, a useful
technique to visualize elements in normally colourless animal tissue.
In fluorescence microscopy, structures are visualized using fluorescent dyes as labels. Fluoresence is a
phenomenon where the chemical structure of the dye captures electromagnetic radiation of one wavelength
(the excitation wavelength) and releases it as radiation of another, lower energy wavelength (the emission
wavelength). For example, when light at 488nm (in the blue region of the visible spectrum) falls upon green
fluorescent protein, electrons in the outer orbital of the atoms within the protein are excited to a higher
energy state. When they return to their normal energy state, they emit photons of light at 509nm, which is in
the green region of the visible spectrum (see Figure 5).
In fluorescence microscopy, light of the desired excitation wavelength is shone onto the specimen, exciting the
fluorescent labels. The light emitted is passed up through the optics of the microscope to the eyes or a light
detector. To prevent interference from reflected excitation light entering the optics, a dichroic mirror is used,
which allows the emitted light to pass through, but not the excitation light. Further sensitivity can be achieved
using emission filters, which only allows light of the desired emission wavelength to pass through. When light
is shone down through the entire specimen, this is known as epifluorescence. Fluorescence microscopes have
a number of adjustable filters, so that a range of excitation and emission wavelengths can be selected (see
Figure 6).

Page 14
488nm 509nm
e- e-
GFP GFP
Figure 5 - Excitation of GFP by light at 488nm (blue) to create emission at 509nm (green)
Eyepiece lens
Excitation
Emission Filter Filter
White Light
Dichroic
Mirror
Objective
Lens
Specimen
Figure 6 - Schematic of an Epifluorescence Microscope

Page 15
Due to the arrangement of excitation and emission filters, fluorescence microscopes can only image a single
fluorophore at a time. Therefore, in specimens which have multiple fluorophores, each with different
excitation and emission wavelengths, multiple images must be taken. The microscope is set up for each
fluorophore and an image taken before it is set up for the next fluorophore. The collected images, called
channels, are then combined to create the final image. The light emitted by the fluorophores is often very
weak, or at a wavelength which is difficult to detect with the eyes (the 647nm fluorophore used in this
investigation, for example, cannot be seen by the human eye). Therefore, the digital cameras used in
fluorescence microscopes take the images in grey scale as full colour detectors are less sensitive. Before the
channels are combined, each is assigned a false colour which corresponds to the colour of the light emitted
(see Figure 7).
Blue Channel Green Channel Red Channel

(DAPI/DNA) (GFP) (Tubulin)
False colour False colour False colour

Blue Green Red
Merge
Figure 7 - Combining channels to make a three-colour image

Page 16
Retaining the three channels in the final image can be useful for scientists, as the grayscale images make it
easier to see finer detail than the colour ones, and comparing separate channels makes it easier to detect
different fluorophores co-localised or situated close to one another. As a result, when presenting their results
in lectures or scientific papers, scientists often present the three channels as grayscale images, followed by the
combined colour image (see Figure 8).
Green Red Blue

(GFP) (Tubulin) (pPLK1) Merge
Figure 8 - Demonstration of presentation of fluorescence microscopy results
A limitation of using epifluorescence microscopy is the difficulty in determining whether a combined signal is
the result of two fluorophores in contact with each other (co-localisation). For example, if a green flurophore
and a red fluorophore appear close to each other in the combined image, they appear yellow. This could be
due to the structures they are attached to being in close physical contact, an important consideration when
using fluorescence microscopy to determine interactions between proteins. However cells are three
dimensional objects with a reasonably large depth of field, so the two fluorophores may not actually be close
to each other, but actually overlapping through the depth of the cell (see Figure 9).
One solution to this problem is the use of confocal microscopy. In this method, background fluorescence is
limited by the use of a pinhole aperture (at the expense of signal intensity, resulting in longer exposure times).
In addition, the excitation of the fluorophores is done by tightly focused lasers which scan through the
specimen. Therefore, only a particular portion of the specimen is exposed to the excitation wavelength and a
virtual section is created. If structures are co-localised, they will appear on the same section, whereas if they
are distant but overlapping, only one will appear on the section. Another benefit to confocal microscopy is that
sequential sections can be stacked on top of one another to create a three dimensional image (a “Z stack”).
Due to the lack of background fluorescence, confocal images appear clearer than epifluorescence images (see
Figure 10).

Page 17
Co-localisation
Combined signal
appears yellow
Distant but
overlapping
Overlapping signal
appears yellow
Figure 9 - Co-Localisation and overlapping signals
a) b)
Figure 10 - Comparison of epifluorescence and confocal microscopy.

a) HeLa cells imaged using epifluorescence – Blue is DAPI (staining DNA), red is tubulin, green is GFP
b) HeLa H2B GFP cells imaged using confocal microscopy – Blue is pPLK1, red is tubulin, green is GFP
(localized to nucleus)

Page 18
Experimental Protocols
How to Use this Manual
Throughout this section you will see a series of icons which represent what you should do at each point. These
icons are:
 Write down a result or perform a calculation.
Prepare a reaction tube.
 Incubate your samples.
When you are asked to deliver a set volume, the text will be given a colour representing the colour of the
micropipette used:
e.g. 750µL Use the blue P1000 micropipette (200-1000µL)
100µL Use the strong yellow P200 micropipette (20-200µL)
15µL Use the pale yellow P20 micropipette (2-20µL)
2µL Use the orange P2 micropipette (0.1-2µL)
Preparation of Culture Plates
Prior to this laboratory session cells have been seeded into 6 well culture plates with poly-L-lysine coated
coverslips on the bottom of each well. The cells have been allowed to attach to the coverslips and grow
overnight. The culture medium was removed from each well and the cells were washed three times by
replacing the medium with phosphate buffer saline (PBS - a solution which does not put the cells under
osmotic stress) and removing. The cells were then fixed in a solution of 4% paraformaldehyde in PBS for 20
minutes, before being washed three times again in PBS.
You will be working in pairs. Three pairs will work with one culture plate, allowing for two coverslips per pair.
Blocking and Permeablisation
Add 3mL of Block / Permeablisation solution (1X PBS containing 1% BSA and 0.1% Triton X100) to each
of the remaining wells
 Incubate the coverslips at room temperature for 15 minutes

Page 19
Treatment with Antibodies
The primary antibodies need to be diluted first in Blocking Solution (1XPBS containing 1% BSA). Prepare the
following antibody dilutions :
Volume Volume
1° Antibody Source
Needed Block. Soln.
α Tubulin Rabbit 1µL
500µL
α Complex IV Mouse 1µL
In order to account for background fluorescence, a negative control should be prepared for the primary
antibodies. This coverslip should be placed on a drop of blocking solution rather than diluted antibody.
 Set up 50µL spots of diluted primary antibody on a piece of Parafilm, one for each coverslip, as well as
a 50µL drop of blocking solution for the negative control
 Place each coverslip CELL SIDE DOWN on separate spots on the Parafilm
 Incubate the coverslips under a foil cover for 1 hour at room temperature
 Set up three 100µL spots of PBS for each coverslip on a separate piece of Parafilm
 Wash the coverslips by placing each CELL SIDE DOWN on each spot and transferring to the next
Prepare the secondary antibody solutions by diluting them in Blocking Solution (PBS containing 1%
BSA) as indicated below :
Volume Volume
Coverslip 2° Antibody
Needed Block. Soln.
α Tubulin α Rabbit 647 1µL
300µL
α Complex IV α Mouse 405 1µL
 Set up 50µL spots of the secondary antibody solutions on Parafilm and place each coverslip CELL SIDE
DOWN on separate spots. In this case, the negative control coverslip should be placed on a spot of
diluted secondary antibodies
 Incubate the coverslips under aluminium foil for one hour at room temperature
 Wash the coverslips three times in PBS as before
 Rinse off the PBS by dipping the coverslip in water
 Prepare 10µL drops of Mowiol mounting medium on microscope slides

Page 20
 Mount the coverslips CELL SIDE DOWN on the drops of Mowiol
 Place the slides under a stream of hot air from a floor heater for 15 minutes.
Imaging Using Epifluorescence Microscopy
Observe the coverslips using the fluorescence microscope as directed by you tutor. Most of the cells visible
should be in interphase, allowing you to see a normal nucleus and distribution of tubulin and mitochondria.
Due to the poly-L-lysine coating on the microscope, you should see some cells at different stages of mitosis.
The three components visualized should be :
 The Nucleus / Chromosomes – The nucleus will appear as a pale green blob with a distinct boundary
around its outside. Inside the nucleus should be some unstained regions which resemble bubbles –
these are the nucleoli and represent centres of RNA synthesis. During mitosis, the chromosomes
condense and the green signal will become stronger. In prophase and prometaphase, you will see the
DNA clump together in bright green bundles (the chromosomes) while the clear boundary around the
outside of the nucleus will disappear. In cells in metaphase, the chromosomes line up as a line across
the middle of the mitotic spindle (the “metaphase plate”), while in anaphase and telophase, you
should see the chromosomes separate out into separate bundles at opposite ends of the cell.
 Tubulin – tubulin should appear in interphase cells as a diffuse fibrous red material. In mitosis, from
the end of prometaphase, this consolidates into the mitotic spindle – a birdcage like structure with
strands of tubulin radiating out from the ends. The mitotic spindle should start to break down by the
end of telophase.
 Complex IV / Mitochondria – mitochondria should as tiny blue specks throughout the cytoplasm of the
cell – they are actually much smaller than how they are portrayed in diagrams in textbooks.
Things to Look Out For
 The Stages of Mitosis – see if you can get an image of cells at each stage of mitosis
 Aberrant Cells – HeLa cells have been continuously subcultured since 1952. This means that they have
accumulated a lot of mutations, on top of those that led to the cells turning cancerous in the first
place. Observe the diversity of cell shape and size in the cells present, along with other anomalies such
as cells with more than one nucleus.
 Aberrant Mitosis – some of the mutations HeLas have accumulated include changes to the normal
process of mitosis. A more common anomaly is cells dividing in three rather than two – these cells
have a three-pronged metaphase plate (like the Mercedes-Benz symbol) and in anaphase you will see
the chromosomes being pulled in three directions rather than two.
 Where are the Mitochondria ? – what happens to the mitochondria during mitosis ? How are they
parceled out between the two daughter cells ?

Page 21
Appendix A : DNA
 Deoxyribonucleic acid (DNA) is a large molecule which stores the genetic information in organisms. It is
composed of two strands, arranged in a double helix form. Each strand is composed of a chain of
molecules called nucleotides, composed of a phosphate group, a five carbon sugar (pentose) called
deoxyribose and one of four different nitrogen containing bases.
NH 2
N
N
O
O P O- N
N
O O
O Nitrogenous
5' H H
Bases
H H
3' N
NH
O H
-
O P O N
Phosphate N NH 2
Group
O O NH 2
5' H H
H H
3' N
Deoxyribose O H
O P O- O N
O O O
5' H H
H H HN
3'
O H
O P O- O N
O O
5' H H
H H
3'
OH H
Figure 3 – The Structure of a Single Strand of DNA
 Each nucleotide is connected to the next by way of covalent bonding between the phosphate group of
one nucleotide and the third carbon in the deoxyribose ring. This gives the DNA strand a “direction” –
from the 5’ (“five prime”) end to the 3’ (“three prime”) end. By convention, a DNA sequence is always
read from 5’  3’ ends.

Page 22
 DNA nucleotides contain one of four different nitrogenous bases:
Adenine Guanine
Thymine Cytosine
Each of these bases jut off the sugar-phosphate “backbone”. If the double helix of the DNA molecule
can be thought of as a “twisted ladder”, the sugar-phosphate backbones form the “rails”, while the
nitrogenous bases form the “rungs”.
 The two strands of DNA are bound together by hydrogen bonding between the nucleotides. Adenine
always binds to thymine and guanine always binds to cytosine. This means that the two strands of DNA
are complementary. The complementary nature of DNA is allows it to be copied and for genetic
information to be passed on - each strand can act as a template for the construction of its
complementary strand.
 The order of bases along a DNA strand is called the DNA sequence. It is the DNA sequence which
contains the information needed to create proteins through the processes of transcription and
translation.
 Each strand of DNA is anti-parallel. This means that each strand runs in a different direction to the
other – as one travels down the DNA duplex, one strand runs from 5’  3’, while the other runs 3’ 
5’.
 An animation of the structure of DNA can be found at:

http://www.johnkyrk.com/DNAanatomy.html

Page 23
Appendix B : Proteins
 Proteins are large molecules composed of chains of amino

acids. Each amino acid is joined to the next one by peptide
bonds, which form between the acid (containing a carbon
atom) of one amino acid and the amino group (containing a
nitrogen atom) of the next. This means that all of the amino
acids are arranged in the same “direction” in the protein and
that one end of the protein can be designated the “carbon
end” (or C Terminus) and the other end the “nitrogen end” or (N terminus).
 Because a protein consists of many amino acids joined by peptide bonds, the word “peptide” is used to
describe proteins – we may use “peptide chain” to describe the sequence of amino acids,
“oligopeptide” refers to a sequence of a few (e.g. a dozen or so) amino acids, while “polypeptide”
refers to sequences much longer and is equivalent to the term protein.
 Because proteins are so large, we don’t often refer to their actual molecular mass. Instead we use a
unit called a Dalton, which is equivalent to a molecular mass of 100, or the average molecular mass of
an amino acid. Therefore, the size of a protein expressed in Daltons is equivalent to how many amino
acids it contains (e.g. a protein with a size of 244 Daltons is 244 amino acids long).
 There are twenty different amino acids found

in nature. It is different combinations of these
amino acids which affect the structure and
function of the proteins. Amino acids all have
the same basic structure, and only differ by
the chemical structure of a “side chain” which
comes off the central carbon atom. Amino
acids are sometimes referred to as “residues”.
You can find an excellent tutorial (including a

quiz) on amino acids at :
http://www.biology.arizona.edu/biochemistry
/problem_sets/aa/aa.html
 Proteins can be thought of as having a number

of levels of structure – the primary,
secondary, tertiary and sometimes quaternary
structures. A video which briefly describes
each of these can be found at:
http://www.youtube.com/watch?v=lijQ3a8yU
YQ
Image from http://www.genome.gov/DIR/VIP/

Page 24
Structures of the Twenty Amino Acids
Polar Non-polar “Special”
Aspartic Acid Glutamic Acid Lysine Arginine Histidine

(asp or D) (glu or E) (lys or K) (arg or R) (his or H)
Serine Threonine Glutamine Asparagine Tyrosine

(ser or S) (thr or T) (gln or Q) (asn or N) (tyr or Y)
Alanine Valine Leucine Isoleucine Methionine

(ala or A) (val or V) (leu or L) (ile or I) (met or M)
Phenylalanine Tryptophan Glycine Cysteine Proline

(phe or F) (trp or W) (gly or G) (cys or C) (pro or P)

Page 25
 The order of amino acids in a protein is called the primary structure. The peptide bond is a covalent
bond with partial double bond characteristics. This means that the molecules do not rotate around the
peptide bond and the bond remains rigid, giving some structure to the chain. Because covalent bonds
are quite strong, it takes a lot of energy to break up the primary structure of a protein.
 Once a chain of amino acids has been assembled by the ribosome, it tends to fold in on itself. Nitrogen
and oxygen atoms are strongly electronegative, which means that they will attract hydrogen atoms
bound to nearby atoms. The first hydrogen bonds to form tend to occur between the atoms which
make up the peptide bonds. This makes for tight structures within the protein. When the chain coils up
like a corkscrew, an α helix is formed. When the chain folds up in parallel lines, a β pleated sheet is
formed. Both of these components make up the secondary structure of the protein. Whether a region
of the peptide chain produces α helices or β pleated sheet depends largely on the chemical nature of
the amino acids which makes them up.
α Helix
β Pleated Sheet
(from Wikimedia Commons)
 A protein then folds up to give further three-dimensional

structure, based on hydrogen bonds between the side chains
of its amino acids and their interaction with the surrounding
environment. If the side chain of an amino acid is non-polar
(hydrophobic or water repelling) it is pushed to the inside of
the protein molecule by the aqueous environment inside the
cell. Polar or charged side chains tend to be found on the
outside of the protein molecule. This three-dimensional
structure is the tertiary structure of the protein. The tertiary The tertiary structure of a protein,
structure determines the function of the protein and how it showing α helices (red) and β pleated
sheets (gold)
interacts with other substances. (from Wikimedia Commons)
 Hydrogen bonds are quite weak compared to covalent bonds. It does not take much to disturb a
hydrogen bond – gentle heating (e.g. above 60°C), changing the (from:
pH,http://www.org.chemi.e.tu-
the level of salts or the presence
muenchen.de/people/mh/Kdp/kdp.html)
of certain disrupting surfactants can all interfere with the hydrogen bonding which holds the tertiary
structure of proteins together. When this happens, we say that the protein has been denatured. An
example of this is when we heat egg white. The mostly transparent and water soluble proteins which
make up albumen are denatured by the heat, producing the hard, insoluble and opaque substance we
associate with cooked egg. Once a protein has been denatured, it can no longer carry out its normal
function – solubility changes and alterations occur in the tertiary structure which means that the
protein can no longer bind to other substances or catalyse chemical processes.

Page 26
 Sometimes stronger bonds form between regions in the tertiary structure of proteins. Cysteine is a
sulphur containing amino acid. In proteins which contain a lot of this residue, nearby sulphur atoms
may form covalent disulphide bridges. These covalent bonds give a strength to the protein not found
in others. Tough proteins like keratin (found in fingernails and hair) contain a lot of disulphide bridges.
 The protein can be thought of as containing a number of distinct regions called domains. Each domain
either has a particular structure (e.g. a large number of α helices) or a distinct function in the
molecule. For example, while in most proteins, domains with a high proportion of non-polar amino
acids are forced to the inside of the molecule, transmembrane proteins tend to have hydrophobic
domains where these side chains are on the outside. This allows these proteins to embed in and attach
to the non-polar inner part of the cell membrane.
 Some proteins, once assembled and folded, may join together

with other proteins to form larger molecules. This is called the
quaternary structure and is not found in all proteins. If two or
more identical subunits join together, the results are called a
homodimer, homotrimer, homotetramer, etc. If the subunits
are different, they are called heteromers. For example, the
protein haemoglobin is a tetramer consisting of two α chains
and two β chains.
 Other groups may also be attached to proteins to assist in its

function. Lipids may incorporate to form lipoproteins which The quaternary structure of Haemoglobin,
may be associated with the cell membrane or involved in the showing the two pairs of subunits (red and
gold) and the haem group (green)
transport of lipid substances like cholesterol in the aqueous (from: Wikimedia Commons)
environment of the blood. Carbohydrates associate with
proteins to form glycoproteins, important as cell surface proteins involved in cellular recognition and
in structural components like cartilage.
 Proteins with a transport or catalytic role may also incorporate metal ions encased in special molecular
cages. For example, each subunit in the haemoglobin molecule contains a carbon and nitrogen
containing cage called a haeme group which binds to the iron ions. This allows the haemoglobin
molecule to transport molecular oxygen throughout the body in the blood. Such proteins are called
metalloproteins.

Page 27
Protein Biochemistry Links
 Folding@Home – a distributed computer project aimed at investigating how proteins fold. Download
the software and help protein scientists in their research.
http://folding.stanford.edu/
 Folding@Home’s guide to Proteins – excellent summary from amino acids to protein folding. Includes
some great animations.
http://www.stanford.edu/group/pandegroup/folding/education/protein.html
 Introduction to Protein Structure – a presentation by Dr Frank Gorga.

http://webhost.bridgew.edu/fgorga/proteins/default.htm
 Interactive Concepts in Biochemistry – a wide range of molecular biology related interactive activities,
including an amino acid identification game.
http://www.wiley.com/legacy/college/boyer/0470003790/animations/animations.htm

Page 28
Appendix C : Using a Micropipette
When scientists need to accurately and precisely deliver smaller volumes of a liquid, they use a pipette – a
calibrated glass tube into which the liquid is drawn and then released. Glass and plastic pipettes have been
mainstays of chemistry and biology laboratories for decades, and they can be relied upon to dispense volumes
down to 0.1mL.
Molecular biologists frequently use much smaller volumes of liquids in their work, even getting down to 0.1µL
(that’s one ten thousandth of a millilitre, or one ten millionth of a litre!). For such small volumes, they need to
use a micropipette.
Plunger
Micropipettes are called a lot of different names, most
of which are based on the companies which
manufacture. For example, you might hear them called
Tip Eject “Gilsons”, as a large number of these devices used in
Button
laboratories are made by this company. Regardless of
Volume
Adjustment
the manufacturer, micropipettes operate on the same
principle: a plunger is depressed by the thumb and as it
Volume 0 is released, liquid is drawn into a disposable plastic tip.
Readout 5
0
When the plunger is pressed again, the liquid is
dispensed.
Tip Eject
The tips are an important part of the micropipette and
Shaft allow the same device to be used for different samples
(so long as you change your tip between samples)
without washing. They come in a number of different
sizes and colours, depending on the micropipette they
are used with, and the volume to be dispensed.
Tip
Attachment

Page 29
The most commonly used tips are:
 Large Blue – 200-1000µL

 Small Yellow – 2-200µL
 Small White - <2µL
They are loaded into tip boxes which are often sterilised to prevent contamination. For this reason tip boxes
should be kept closed if they are not in use. Tips are loaded onto the end of the micropipette by pushing the
end of the device into the tip and giving two sharp taps. Once used, tips are ejected into a sharps disposal bin
using the tip eject button. Never touch the tip with your fingers, as this poses a contamination risk.
The plunger can rest in any one of three positions:
Position 1 is where the Position 2 is reached by pushing Position 3 is reached by pushing

pipette is at rest down on the plunger until down from Position 2
resistance is met
Each of these positions plays an important part in the proper use of the micropipette.

Page 30
To Draw Up Liquid:
 Hold the micropipette with the thumb resting on the plunger and the fingers curled around the upper
body.
Push down with the thumb Keeping the plunger at the Steadily release pressure on
until Position 2 is reached. second position, place the tip the plunger and allow it to
attached to the end of the return to Position 1. Do this
micropipette beneath the surface carefully, particularly with
of the liquid to be drawn up. Try large volumes, as the liquid
not to push right to the bottom may shoot up into the tip and
(especially if you are removing the body of the micropipette.
supernatant from a centrifuged If bubbles appear in the tip,
pellet), but ensure that the tip is return the liquid to the
far enough below the surface of container by pushing down to
the liquid that no air is drawn up. Position 3 and start again (you
may need to change to a dry
tip).

Page 31
To Dispense Liquid:
 Hold the micropipette so that the end of the tip containing tip is inside the vessel you want to deliver it
to. When delivering smaller volumes into another liquid, you may need to put the end of the tip
beneath the surface of the liquid (remember to change the tip afterwards if you do this to save
contaminating stock). For smaller volumes you may also need to hold the tip against the side of the
container.
Push the plunger down to To remove the last drop of Release the plunger and allow
Position 2. If you wish to mix liquid from the tip, push down it to return to Position 1
two liquids together or to Position 3. If delivering into a
resuspend a centrifuged pellet, liquid, remove the tip from the
release to Position 1 and push liquid before releasing the
to Position 2 a few times to plunger
draw up and expel the mixed
liquids

Page 32
Changing the Volume:
Some micropipettes deliver fixed volumes, however the majority are adjustable. Each brand uses a slightly
different method to do this – Gilsons have an adjustable wheel, others have a locking mechanism and turning
the plunger adjusts the volume. All have a readout which tells you how much is being delivered and a range of
volumes which can be dispensed. Trying to dipense less than the lower value of the range will result in
inaccurate measurements. Trying to dispense over the upper range will completely fill the tip and allow liquid
to enter the body of the pipette. Do not overwind the volume adjustment, as this affects the calibration of the
micropipette.
The way to interpret the readout depends on the micropipette used:
In a 200-1000µL micropipette (e.g. a Gilson

P1000) the first red digit is thousands of µL (it
0 should never go past 1), the middle digit is
P 3 hundreds, while the third is tens. Therefore
1000
5 1000µL would read as 100, while 350µL would
read as 035.
In a 20-200µL micropipette (e.g. a Gilson P200)

the first digit is hundreds of µL (it should never
0 go past 2), the second is tens and the third is
P units. Therefore, 200µL would read as 200,
9
200 while 95µL would read as 095.
5
In a 2-20µL micropipette (e.g. a Gilson P20) the

first digit is tens of µL (it should never go past
0 2), the second is units and the third red digit is
P 2 tenths. Therefore 20µL would read as 200,
20 while 2.5µL would read a 025.
5
In a 0.2-2µL micropipette (e.g. a Gilson P2) the

first digit is units of µL (it should never go past
0 2), the second red digit is tenths and the third
P2 5 red digit is hundredths. Therefore, 2µL would
0 read as 200, while 0.5µL would read as 050.

Page 33
Appendix B : Glossary of Terms
Amino Acid – a small organic molecule which has a component which acts as a base (an amine group –NH2) and one
which acts as an acid (carboxyl group –COOH). Amino acids are the building blocks of proteins. Hanging off the central
carbon atom is a side chain (often given the abbreviation R) which differentiates one amino acid from another. There are
approximately 20 different amino acids which are used to make proteins.
Antibody – a specialised protein molecule produced as part of the immune response which binds specifically to other
molecules.
Antibiotic – a compound which interferes with the growth of micro-organisms, particularly bacteria.
Antigen – an agent to which an antibody binds.
Bacterium – a microorganism with a cell wall but which lacks membrane-bound organelles.
Bases – the four organic molecules which are found in nucleotides. The bases found in DNA are adenine, thymine,
guanine and cytosine. In RNA, thymine is replaced by uracil.
Biochemistry – the study of the chemistry of living things.
Biomolecule – a complex organic compound which is made as the result of a biological process. Also called
macromolecules, because most are quite large.
BSA – bovine serum albumin – a protein solution derived from the blood of cows.
Buffer – a compound which helps to keep the pH of a solution stable and constant.
Cancer – a condition characterized by abnormal cell growth and multiplication, as well as migration of affected cells
throughout the body.
Cell – the basic unit of all living things. Cells are metabolically active membrane bound bodies capable of reproduction.
Cell Biology – the study of processes which cells use to survive.
Cell Cycle – the progression of stages which a cell passes through in its growth and development. It consists of G1 (Gap 1)
phase, where organelles are produced and the cell starts to increase in size, S (Synthesis) phase, where DNA is replicated
so that each daughter cell has a complete copy of the genome, G2 (Gap 2) phase, where the cell checks that all is in order
for division, and M (Mitosis) phase, where the chromosomes are separated (mitosis) and the cell divides into two
daughter cells (cytokinesis). Following M phase, cells return to G1 phase should they need to divide again. Most cells go
from G1 phase into G0 phase, where they carry out their normal cellular functions, as most cells do not need to
constantly divide. Changes to the cell cycle can lead to a situation where the cells are constantly dividing, a state which
may progress to cancer. An understanding of the processes which control the cell cycle can lead to ways to treat cancer,
either by stopping the cell cycles of cancerous cells, or preventing cells from turning cancerous in the first place.
Cell Membrane – the outer boundary of the cell. It is composed of a double layer of phospholipids molecules arranged so
that the water soluble ends face outwards while the water insoluble fatty ends face inwards. Embedded in this bilayer
are various proteins involved in recognition of other substances and transfer of materials across the membrane.
Cell Line – an immortal population of cells used in research derived from a tumour.
Co-localisation – the close proximity of two structures.

Page 34
Complex IV - cytochrome C oxidase – a protein found in the membranes of mitochondria which carries out part of the
electron transport chain in cellular respiration. Complex IV can be used as a marker to demonstrate mitochondria in
immunofluorescence.
Confocal Microscopy – fluorescence microscopy in which the resolution is improved by focusing laser light of the
excitation wavelength onto the specimen and passing the emitted light through a pinhole.
Chromogen – a compound which changes colour, after undergoing a chemical reaction.
Chromosome – A length of DNA. Human cells have 46 linear chromosomes, while bacteria have a single circular
chromosome.
Culture – the practice of growing cells by providing them with the right temperature and nutrient requirements.
Cytochemistry – the use of stains and dyes to demonstrate structures within cells.
Denaturation – a disruption to the structure of a macromolecule caused by the breaking of hydrogen bonds. In DNA,
denaturation results in the separation of the DNA strands, while in proteins it results in the loss of secondary and tertiary
structure.
Diabetes – a metabolic condition characterised by a disturbance in the normal control of glucose metabolism. The
balance of glucose between the inside of the cell and the spaces outside (e.g. the blood) is maintained in part by the
hormone insulin. In diabetes, this balance is disturbed, either by abnormal or insufficient production of insulin (Type I
Diabetes) or a failure of cells to properly respond to insulin (Type II Diabetes). A third type of diabetes (Gestational
Diabetes) occurs during pregnancy.
Dichroic Mirror – a structure which allows some wavelengths of light to pass through while reflecting others.
Dilution – reducing the concentration of a solution by adding more solvent.
DNA – deoxyribonucleic acid – the biomolecule which stores the genetic information in most living things. DNA consists of
two strands of deoxynucleotides linked by phosphodiester bonds. The bases in the two nucleotide strands bind in
complementary pairs (adenine to thymine, cytosine to guanine) through hydrogen bonds. This gives the molecule the
appearance of a twisted ladder, with the sugar-phosphate chains forming the runners and the base pairs forming the
rungs. The sugar in the nucleotides which make up DNA is deoxyribose.
Electromagnetic Radiation – a form of radiation which includes radio waves, microwaves, infra-red radiation, visible light,
ultraviolet radiation, X-rays and gamma rays.
Emission – in fluorescence, the wavelength emitted by a fluorophore following excitation.
Enzyme – a protein which acts as a biological catalyst – it speeds along reactions which would normally be too slow to be
useful.
Epifluorescence – fluorescence observed after shining light of the appropriate excitation wavelength onto or through a
specimen.
Epitope – the portion of a protein which generates an immune response.
Eukaryote – an organism whose cells containing membrane-bound organelles (eg. protists, fungi, plants and animals).
Excitation – in fluorescence, the wavelength needed to raise the electrons of a fluorophore to a higher energy state.

Page 35
Filter – in optics, a structure which only allows a limited range of wavelengths of light to pass through.
Fixation – a chemical treatment which forms crosslinks between peptide chains in proteins and prevents degradation.
Fluorophore – a compound, usually attached to another molecule, which fluoresces.
Fluorescence – the emission of visible light by a substance after it has been excited by light of a higher energy.
Fungi - a group of eukaryotic organisms characterised by the presence of chitin in their cell walls. Fungi may be unicellular
(yeasts) or multicellular (filamentous fungi). Fungi may be pathogenic, particularly in individuals with a compromised
immune system.
Gene – a small section of DNA which contains the information used to produce a protein, or which controls and regulates
the expression of other genes.
GFP – Green fluorescent protein – a protein derived from a jellyfish which emits green light when irradiated with
ultraviolet light. GFP is commonly used as a tag to localise substances in molecular biology.
HeLa Cells – a line of immortal cells grown in culture which were derived from a cervical tumour removed from a woman
named Henrietta Lacks in 1951. They are one of the more widely used mammalian cell lines.
Histochemistry – a laboratory discipline which involves studying thin sections of tissue which have been stained using
various dyes.
Histone – a family of proteins intimately associated with DNA in the nucleus. DNA wraps around histones to form
nucleosomes. This process assists in chromosomal packing and gene regulation.
Human papillomavirus (HPV) – a virus which infects the cells of the skin and genital mucosa which is implicated in the
development of warts and some cancers.
Hyperproliferation – an abnormal increase in the rate of cell reproduction.
Immunity - protection from a disease causing agent.
Immunofluorescence – a technique in which a cellular component is localised using an antibody attached to a fluorescent
label
Immunohistochemistry – a technique in which enzyme labeled antibodies are used to demonstrate the presence of
proteins and other structures in sections of tissue. Addition of a chromogenic substrate which creates a coloured
precipitate in the presence of the enzyme label indicates the presence of the target molecule.
Incubation – a waiting period, to allow a reaction time to take place, or organisms time to grow and multiply.
In Vitro – “in glass” – experiments performed outside a living organism.
In Vivo – “in life” – experiments performed inside a living organism.
Isotonic – describes a solution in which the concentration of solutes is equal to that found in cells.
Lipid – biomolecules which are generally insoluble in water. Lipids are classified as fats (solid at room temperature) or oils
(liquid at room temperature). Lipids have a role in storage of energy, as well as structural roles (lipids make up the bulk of
cell membranes).

Page 36
Lymphocyte – a type of white blood cell involved in the immune response. Lymphocytes are divided into a number of sub-
types : Natural Killer (NK) cells, T Cells (comprising T Helper (TH) cells and Cytotoxic (TC) cells) and B cells.
Medium – a combination of salts and nutrients dissolved in a liquid (broth) or semi-solid material (plate) in which cells
are grown.
Micropipette – a device used to accurately and precisely deliver small quantities (<1mL) of liquid.
Mitochondrion – a sub-cellular organelle which carries out cellular respiration in eukaryotic cells.
Mitosis – the process by which eukaryotic cells distribute chromosomes during cell division. Mitosis consists of prophase,
prometaphase, metaphase, anaphase and telophase. The separation of the cells themselves is called cytokinesis.
Molecular Biology – the study of how chemical processes contribute to living systems. Molecular biology concentrates
largely on the nature of DNA and proteins.
Mutation – any change to the normal DNA sequence.
Nucleic Acid – a biomolecule consisting of a chain of nucleotides connected by phosphodiester bonds. DNA and RNA are
nucleic acids.
Nucleolus – a body found within the nucleus of cells involved in the manufacture of ribosomes.
Nucleoside – a combination of one of the nitrogenous bases (adenine, guanine, thymine, cytosine or uracil) and a five
carbon (pentose) sugar – deoxyribose in DNA or ribose in RNA.
Nucleotide – a nucleoside joined to a phosphate (PO4) group. Nucleotides make up nucleic acids.
Nucleus – a membrane bound body inside the cells of eukaryotes which contains the chromosomal DNA.
Organelle – a sub-cellular component which carries out a particular task.
Osmosis - the diffusion of water across the cell membrane.
Pathogen – an agent which can cause disease.
Peptide – a length of amino acids joined by peptide bonds.
pH – the degree of acidity (low pH) or alkalinity (high pH) of a solution.
Plasma Cell – a cell derived from B lymphocytes which generates antibodies.
Protein – a biomolecule consisting of polypeptide chains folded up into three dimensional forms. Proteins play many roles
in organisms, including being the building blocks of cellular structures, control and regulation of chemical reactions
(enzymes), recognition and communication between cells (receptors and hormones) and defense (antibodies).
Rheumatoid Arthritis – an autoimmune disease which results from the body’s immune system attacking the cartilage
tissue in the joints.
Ribosome - a body within the cell composed of protein and ribosomal RNA which carries out translation.
Serum – the liquid (ie. non-cellular) component of clotted blood. Serum is plasma with the clotting factors removed.

Page 37
Stock Solution – a concentrated solution used to store reagents. Stock solutions are usually made to be a certain number
of times more concentrated that the working solutions and so must be diluted by the factor to create the working
solution. eg. 50X stock must be diluted 1 in 50 before it can be used.
Tissue – a collection of cells which work together to perform a function in the body.
Translation – the process of creating a protein following information encoded in a messenger RNA molecule.
Tubulin – a protein which forms a major component of the cytoskeleton of the cell. Because it is found in relatively
constant concentrations in most cells, tubulin is used as a loading control in SDS-PAGE and western blotting.
Tumour – a mass of tissue, often consisting of abnormal or undifferentiated cells. Tumours may be considered as benign
or malignant.
Vaccine - a treatment aimed at artificially stimulating an immune response against a specific agent.
Vacuole – a membrane bound body found within cells used for storage.
Wavelength – the distance between two peaks in electromagnetic radiation. What we perceive as “colour” in the visible
spectrum of light is the wavelength of that light.
Working Solution – the solution which is used in a chemical solution. Working solutions may be made up fresh or diluted
from stock solutions. They are normally given the name “1X” to differentiate them from their stock solutions.
Z Stack – a virtual three dimensional image created by stacking together sequential confocal scans through a specimen.

Page 38

Introduction To Cell & Molecular Biology Techniques: Immunofluorescence

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Introduction To Cell & Molecular Biology Techniques: Immunofluorescence

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Introduction to Cell & Molecular Biology

DAPI (DNA) GFP γ Tubulin

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

What is Cell Biology ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Common Cell Biology Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Theoretical Basis of the Project

Tissue Culture and Cancer Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Preparation of Cells for Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Antibodies and Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

How to Use this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Preparation of Culture Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Blocking and Permeablisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Treatment with Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Imaging Using Epifluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

What to Look Out For . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Appendix C : Using a Micropipette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Appendix D : Glossary of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

What is Cell Biology ?

Common Cell Biology Techniques

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Fluorescence Microscopy – uses fluorescent materials to indicate structures in a specimen.

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Theoretical Basis of the Project

Tissue Culture and Cancer Cell Lines

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Preparation of Cells for Immunofluorescence

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Fixed Cell Permeablisation

Figure 1 - Fate of unfixed and fixed cells following permeablisation

Antibodies and Immunofluorescence

Antibodies are large, complex proteins made by Light Chain

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Treatment with labeled antibody

Antigen Present Antigen Absent

UV Light Unbound antibody washed away UV Light

Antigen Present Antigen Absent

Fluorescence observed where antigen is located

Figure 3 - Principal of Direct Immunofluorescence

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Treatment with unlabelled primary antibody

Antigen Present Antigen Absent

Unbound antibody washed away

Antigen Present Antigen Absent

Treatment with unlabelled secondary antibody

Figure 4 - Principle of Indirect Immunofluorescence

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Unbound secondary antibody washed away

Antigen Present Antigen Absent

Fluorescence observed wherever antigen is located

Figure 4 - Principle of Indirect Immunofluorescence (Continued)

In this workshop, you will be demonstrating three components of the cells :

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

 Complex IV (or Cytochrome C Oxidase) – Complex IV is a large transmembrane protein composed of 13

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Figure 6 - Schematic of an Epifluorescence Microscope

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Blue Channel Green Channel Red Channel

False colour False colour False colour

Figure 7 - Combining channels to make a three-colour image

© 2014 State of Queensland (Department of Education, Training and Employment http://www.di.uq.edu.au/SPARQ-ed

Green Red Blue