HISTOLOGY (LECTURE)
1D HISTOLOGY & ITS METHODS OF STUDY
Dr. LASAM | August 29, 2017
H-01
Topic Outline
I. DEFINITON OF HISTOLOGY
II. PREPARATION OF TISSUE FOR STUDY
a. Fixation
b. Decalcification
c. Dehydration
d. Clearing
e. Infiltration
f. Embedding
g. /Sectioning
h. Staining
III.LIGHT MICROSCOPY
a. Bright-field Microscopy
-definition
-parts and functions
b. Fluorescence Microscopy
c. Phase-contrast Microscopy
d. Confocal Microscopy
e. Polarizing Microscopy
IV. ELECTRON MICROSCOPY
a. Transmission Electron Microscopy
b. Scanning Microscopy
HISTOLOGY
− Study of the tissues
− The Greek root histo can be translated as
either "tissue" or "web"
− aspect of tissue biology with the focus on
how cell structure and arrangement
optimize functions specific to each organs
− advances in molecular biology, physiology,
immunology, and pathology are essential for
a better knowledge of tissue biology.
PREPARATION OF TISSUE FOR STUDY
The most common procedure used in
histologic research is the preparation of tissue
slices or “sections” that can be examined visually
with transmitted light.
A. Fixation
-preserving fresh tissue for examination
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-fixatives have the property of forming
cross-links between proteins
-widely used fixative for light microscopy is
formalin, a buffered isotonic solution of 37%
formaldehyde.
• Primary Aim: To preserve the morphologic and
chemical integrity of the cell as life-like manner
as possible.
• Secondary aim: To harden and protect the
tissue from the trauma of further handling.
Practical considerations of Fixation:
-speed
-duration of fixation
-penetration (1mm/hr)
-volume of fixative (fixative-to-tissue ratio 20:1)
B. Decalcification
-more concentrated acid solutions decalcify
bone more rapidly but are more harmful to the
tissues
-high concentrations and greater amount of
fluid will increase the speed of the process
-dense bone tissues usually require up to 14
days or longer in order to complete the process.
C. Dehydration
- to remove the fixative and water from the
tissues and replacing them with dehydrating fluid
-generally used in increasing strengths (all
the aqueous tissue fluids are removed but with the
little disruption to the tissue due to diffusion
currents)
• Ethanol- for routine dehydration of
tissues
• Methyl alcohol-employed for blood and
tissue films
• Butyl alcohol- for plant and animal
microtechniques
D. Clearing
-removal of alcohol
-should be miscible with BOTH the
dehydrating fluid and the embedding medium
-clearing agent gives the tissue a
translucent appearance
 P-01 Histology and Its Methods of Study
• Xylene- most common and most rapid
clearing agent.
E. Infiltration
-the tissue is then placed in melted paraffin
until it becomes completely infiltrated with this
substance.
• Paraffin- simplest/most common
-not recommended for fatty tissues
• 55-60ºC -Temperature of paraffin oven
(paraffin oven must be maintained at a
temperature 2-5ºC above the melting
point of the paraffin wax)
F. Embedding
-infiltrated tissue is placed into a precisely
arranged position in a mold containing a medium
which is then allowed to solidify
G. Sectioning
-parrafin block is cut into uniformly this
slices to facilitate studies under the microscope
3-10um = for routine histologic sections
10-15um = frozen section
Less than 1um=electron microscopy
Microtome - is used for sectioning paraffin-
embedded tissues for light microscopy. The
trimmed tissue specimen is mounted in the paraffin
block holder, and each turn of the drive wheel by
the histologist advances the holder a controlled
distance, generally from 1 to 10 μm. After each
forward move, the tissue block passes over the
steel knife edge and a section is cut at a thickness
equal to the distance the block advanced. The
paraffin sections are placed on glass slides and
allowed to adhere, deparaffinized, and stained for
light microscope study.
H. Staining
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-most cells and extracellular material are
completely, and to be studied microscopically the
tissue sections must be stained.
-cell components such as nucleic acids with
a negative charge (anionic) have an affinity for
basic dyes and are termed basophilic.
-cationic components such as proteins stain
more readily with acidic dyes and are termed
acidophilic.
Examples of Basic dyes:
Toluidine blue
Alcian Blue
Methylene Blue
-glycosaminoglycan, acid glycoprotein, nucleic
acids (reacts with basic dyes because of the acids
in their composition)
Examples of Acid dyes:
Eosin
Orange G
Acid Fuchsin
-mitochondria, secretory granules, collagen
• Hematoxylin – stains DNA of the cell
nucleus and other acidic structures such
as RNA-rich proteins of the cytoplasm
and matrix of the cartilage producing a
DARK BLUE or PURPLE color.
• Eosin - stains other cytoplasmic
structure and collagen PINK
-acts as counterstain in H&E stain
• Periodic Acid Schiff- utilizes the hexose
rings of polysaccharides and other
carbohydrate-rich tissue structures and
stains such macromolecules distinctly
PURPLE or MAGENTA
• Sudan black – lipid soluble dye
-useful in diagnosis of metabolic
diseases that involve intracellular
accumulations of cholesterol,
phospholipids or glycolipids.
MEMBRANE PROTEINS
LIGHT MICROSCOPY
Conventional bright-field microscopy, as
well as more specialized applications like
fluorescence, phase-contrast, confocal, and
polarizing microscopy, are all based on the
 P-01 Histology and Its Methods of Study
interaction of light with tissue components and are
used to reveal and study tissue features.
A. Bright – field Microscopy
-stained tissue is examined with ordinary
light passing through the preparation
• Resolving Power - defined as the smallest
distance between two structures at which
they can be seen as separate objects.
• Virtual microscopy - used for study of bright-
field microscopic preparations, involves the
conversion of a stained tissue preparation to
high-resolution digital images and permits
study of tissues using a computer or other
digital device, without an actual stained
slide or a microscope.
PARTS AND FUNCTIONS OF A BRIGHT –
FIELD MICROSCOPE
• Eyepiece or ocular lens - magnify this image
another X10 and project it to the viewer,
yielding a total magnification of X40, X100, or
X400.
• Tube: connects the eyepiece to the objective
lenses.
• Revolving nosepiece - also known as the
Turret. Revolving nosepiece has holders for the
different objective lenses.
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• Objective lenses - enlarge and project the
illuminated image of the object toward the
eyepiece. Interchangeable objectives with
different magnifications routinely used in
histology include X4 for observing a large area
(field) of the tissue at low magnification; X10 for
medium magnification of a smaller field; and
X40 for high magnification of more detailed
areas.
• Diaphragm - regulates the amount of light on
the specimen
• Coarse adjustment knob - moves the stage up
and down. Used to find a specimen when using
the low power objective
• Fine adjustment knob - moves the body tube for
focusing the high power lens.
• Arm -used to support the microscope when
carried
• Stage - the platform that is flat used for placing
the slides under observation.
• Stage clip: Stage clips hold the slides in proper
place.
• Condenser -collects and focuses a cone of light
that illuminates the tissue slide on the stage.
• Base - supports the microscope
• Power switch - turns the light on and off
B. Fluorescence Microscopy
-uses ultraviolet light, under which only
fluorescent molecules are visible, allowing
localization of fluorescent probes which can
be much more specific than routine stains.
• Fluorescence - when certain cellular
substances are irradiated by light of a
proper wavelength, they emit light with a
longer wavelength
• Acridine orange – a fluorescent stain which
binds both DNA and RNA
C. Phase-contrast Microscopy
-uses the differences in refractive index of
various natural cell and tissue components
to produce an image without staining,
allowing observation of living cells.
-based on the principle that light changes its
speed when passing through cellular and
 P-01 Histology and Its Methods of Study
extracellular structures with different refractive
indices.
D. Confocal Microscopy
-involves scanning the specimen at
successive focal planes with a focused light
beam, often from a laser, and produces a
3D reconstruction from the images.
E. Polarizing Microcopy
-allows the recognition of stained or
unstained structures made of highly
organized subunits
-produces an image only of material having
repetitive, periodic macromolecular
structure; features without such structure
are not seen.
• Birefringence - ability to rotate the direction
of vibration of polarized light
AUTORADIOGRAPHY
MICROSCOPIC AUTORADIOGRAPHY
-is a method of localizing newly synthesized
macromolecules in cells or tissue sections.
-Radioactively labeled metabolites
(nucleotides, amino acids, sugars) provided
to the living cells are incorporated into
specific macromolecules (DNA, RNA,
protein, glycoproteins, and polysaccharides)
and emit weak radiation that is restricted to
those regions where the molecules are
located.
. ■Dehydrogenases, which transfer
CELL AND TISSUE CULTURE
Cell culture- allows the direct observation of cellular
behavior under a phase-contrast microscope and
many experiments technically impossible to
perform in the intact animal can be accomplished in
vitro.
• primary cell cultures- preparations in
which cells to be cultured are dispersed
mechanically or enzymatically from a tissue
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or organ and placed with sterile procedures
in a clear dish to which they adhere, usually
as a single layer
• Some cells can be maintained in vitro for
long periods because they become
immortalized and constitute a permanent
cell line.
• Transformation
- the genetic alteration of a cell resulting
from the direct uptake and incorporation of
exogenous genetic material from its
surroundings through the cell membrane.
-alteration on genetically programmed life
span of cells that promote cell immortality
-are similar to the initial changes in a normal
cell’s becoming a cancer
ENZYME HISTOCHEMISTRY
ENZYME HISTOCHEMISTRY
-is a method for localizing cellular structures
using a specific enzymatic activity present in
those structures.
Pointers to remember for enzyme histochemistry:
(1) tissue sections are immersed in a solution
containing the substrate, a substance which an
enzyme acts, of the enzyme to be localized
(2) the enzyme is allowed to act on its substrate;
(3) the section is then put in contact with a marker
compound that reacts with a product of the
enzymatic action on the substrate
(4) the final product from the marker, which must
be insoluble and visible by light or electron
microscopy, precipitates over the site of the
enzymes, identifying their location. Y
Examples of enzymes that can be detected
histochemically:
include the following:
■Phosphatases, which remove phosphate
groups from macromolecules
hydrogen ions from one substrate to
another, such as many enzymes of the citric
acid (Krebs) cycle, allowing histochemical
identification of such enzymes in
mitochondria.
■Peroxidase, which promotes the oxidation
of substrates with the transfer of hydrogen
ions to hydrogen peroxide.
VISUALIZING SPECIFIC MOLECULES
 P-01 Histology and Its Methods of Study
-A specific macromolecule present in a tissue using labeled complementary DNA (cDNA)
section may also be identified by using tagged probes
compounds or macromolecules that bind
specifically with the molecule of interest.
Examples of molecules that interact specifically
with other molecules include the following:
■ Phalloidin, a compound extracted from INTERPRETATION OF STRUCTURES IN
mushroom, Amanita phalloides, interacts TISSUE SECTIONS
strongly with the actin protein of • Many steps in tissue processing, slide
microfilaments. preparation, and staining can
■ Protein A, purified from Staphylococcus introduce minor artifacts such as spaces
aureus bacteria, binds to the Fc region of and precipitates that are not normally
antibody molecules, and can therefore be present in the living tissue and must be
used to localize naturally occurring or recognized.
applied antibodies bound to cell structures. • Sections of cells or tissues are essentially
■Lectins, glycoproteins derived mainly from 2D planes through 3D structures, and
plant seeds, bind to carbohydrates with high understanding this fact is important for their
affinity and specificity. Different lectins bind correct interpretation and study.
to specific sugars or sequences of sugar
residues, allowing fluorescently labeled
lectins to be used to stain specific
glycoproteins or other macromolecules
bearing specific sequences of sugar
residues.

IMMUNOHISTOCHEMISTRY
- labelled antibodies are routinely used to identify
and localize many specific proteins, not just those
with enzymatic activity that can be demonstrated by
histochemistry.
-based on specific reactions between antigen and
antibodies labeled with visible markers, often
fluorescent compounds or peroxidase for light
microscopy and gold particles for TEM.
• direct immunohistochemistry-process by
which the cell or tissue antigen of interest is
detected by directly binding a labelled
primary antibody specific for that antigen
• Indirect immunohistochemistry- uses an
unlabelled primary antibody that is detected
bound to its antigen with labelled secondary
antibodies.
• The indirect immunohistochemical method
is more commonly used because the added
level of antibody binding amplifies the signal
detected and provides greater technical
flexibility.
• in situ hybridization (ISH)- technique in
which specific gene sequences or mRNAs
of cells can be detected microscopically

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