1B
HISTOLOGY
Dr. Bernal
(Histology & Its Methods of Study)(08.31.2018)
H-01
TOPIC OUTLINE
I. PREPARATION OF TISSUES FOR STUDY
a. Fixation
b. Decalcification
c. Dehydration
d. Clearing
e. Infiltration
f. Embedding
g. Trimming
h. Sectioning
i. Staining
j. Mounting
k. Labelling
II. LIGHT MICROSCOPY
a. Bright-Field Microscopy
b. Fluorescence Microscopy
c. Phase-Contrast Microscopy
d. Confocal Microscopy
e. Polarizing Microscopy
III. ELECTRON MICROSCOPY
a. Transmission Electron Microscopy
b. Scanning Electron Microscopy
IV. AUTORADIOGRAPHY
V. CELL & TISSUE CULTUTRE
VI. ENZYME HISTOCHEMISTRY
VII. VISUALIZING SPECIFIC MOLECULES
a. Immunohistochemistry
b. Hybridization techniques
VIII. INTERPRETATION OF STRUCTURES IN
TISSUE SECTIONS
Histology
 study of the tissues of the body and how these
tissues are arrange to constitute organs.
Two interacting components:
1. Cells
 produce the ECM locally and are in turn
strongly influenced by matrix molecules.
2. Extracellular Matrix (ECM)
 has macromolecules which form complex
structures (i.e. collagen fibrils)
 supports the cells and contain the fluid
transporting nutrients to the cells, and
carrying away their wastes and secretory
products.
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PREPARATION OF TISSUES FOR STUDY
Preparation of tissue slices or ‘’sections’’
 most common procedure used in histologic
research.
Ideal microscopic preparation
 ‘’Preserved’’ so that tissue on the slide has the
same structural features it had in the body.
Fixation
Fixation
 a process to preserve tissue structure and
prevent degradation.
 killing, penetration and hardening of tissues.
 alteration of tissues by stabilizing protein so that
the tissues become resistant to changes (i.e.
degeneration, decomposition, putrefaction and
tissue distortion).
Fixatives
 solutions of chemicals that cross-link proteins and
inactivate degradative enzymes, which preserves
cell and tissue structure.
Examples:
 Formalin – a buffered isotonic solution of 37%
formaldehyde which is widely used for light
microscopy.
 Glutaraldehyde – a fixative used for electron
microscopy that reacts with the amine groups
(NH2) of CHON and also cross-links adjacent
proteins, reinforcing cell and ECM structures.
 Osmium tetroxide – preserves cellular lipids as
well as proteins.
Decalcification
Decalcification
 Process of complete removal of calcium or lime
salts from tissues (bones, teeth, calcified tissues)
after fixation and before infiltration.
5% to 10% Nitric Acid
 Most common and fastest decalcifying agent
 H-01 Histology and Its Methods of Study

temperature between 5 to 10°C above the melting

Dehydration
Dehydration
 Process of removing water from the tissue
following fixation in preparation for wax
impregnation.
 Occurs in a series of increasingly concentrated
alcohol solutions, ending in 100% which removes
all water.
point and then cooled rapidly at -5°C or immersed
in cold water to solidify.
Orientation
 Process by which a tissue is arranged in a precise
position in the mold during embedding, on the
microtome, before cutting and on the slide before
staining.
 Most critical step in embedding

Some dehydrating agents are the following: Paraffin Wax Impregnation

 Ethyl alcohol – most commonly used and for
routine dehydration of tissues
 Acetone – can be a fixative and a dehydrating
agent
 Butyl alcohol – for plant and animal
microtechniques
 Simplest, most common and best embedding
medium used for routine tissue processing
Plastic Resin Embedding
 for electron microscopy and ultra-thin sectioning
for high resolution light microscopy

Clearing Trimming

Clearing/Dealcoholization
 Process wherein alcohol/dehydrating fluid is
removed from the tissue and replaced with a
substance that is miscible with both the
dehydrating fluid and the impregnating or
embedding medium.
Trimming
 process of removing excess wax after embedding
using a knife or a blade to form a truncated
pyramid or a 4 sided prism.
*Tissue should be surrounded by at least 2mm of wax

Xylene
 Most common and most rapid clearing agent Sectioning

Sectioning/Cutting
Infiltration/Impregnation  process wherein a processed tissue is cut into
uniformly thin slices (sections) using microtome
Infiltration/Impregnation to facilitate studies under the microscope.
 Process whereby the clearing agent is completely
removed from the tissue and replaced by a Type of Section Usual Thickness
medium that will completely fill all the tissue Paraffin Section LM: 3-10 micra EM: <1 micra
cavities. Celloidin Section 10 – 15 micra
Frozen Section 10 micra
Paraffin
 Most widely used infiltrating medium. Microtome
 use for sectioning paraffin-embedded tissues for
light microscopy

Embedding Cryostat
 a microtome in a cabinet at subfreezing
Embedding/Casting/Blocking/Molding temperature which is used to section the block
 Paraffin embedded tissues are arranged at the with tissue.
bottom of the mold together with their proper
labels and immersed in melted paraffin at a

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  H-01 Histology and Its Methods of Study

Staining Metal Impregnation

 Solutions of silver salts are used to visualize
Staining certain ECM fibers and specific cellular elements
 Process of applying dyes on the sections to see in nervous tissue.
and study the architectural pattern of the tissue
and physical characteristics of the cell.
LIGHT MICROSCOPY
Basophilic
 Cell components such as nucleic acids with a net Bright-Field Microscopy
negative charge (anionic) have an affinity for
basic dyes.
Bright-Field Microscopy
Examples:
 Uses ordinary light passing through the
 Toluidine blue
preparation and the colors are imparted by tissue
 Alcian blue
staining.
 Methylene blue
 Hematoxylin – stains DNA in the nucleus, RN-rich
Condenser
cytoplasmand matrix of cartilage, producing a
 Focuses light on the object to be studied.
dark blue or purple color.
***Basophilic components of tissues such as DNA,
Objective Lenses
RNA and glycosaminoglycans.
 Enlarge and project the image of the object
toward the observer
Acidophilic
 Cationic components, such as proteins with many
Eyepiece
ionized amino groups, stain more readily with
 Magnifies the image and projects it into the
acidic dyes.
viewer’s retina
Examples:
 Eosin – stains cytoplasmic structures and
Total Magnification
collagen pink; counterstain
 Total magnifying power of the objective lens and
 Orange G
ocular lenses.
 Acid fuchsin
Scanner Objective 4 x 10 = 40x
***Acidophilic components of tissues such as
Low Power Objective 10 x 10 = 100x
mitochondria, secretory granules, and collagen.
High Power Objective 40 x 10 = 400x
Oil Immersion Objective 100 x 10 = 1000x
Hematoxylin and Eosin (H&E)
 Most commonly used stains
Resolving Power
 Smallest distance between two structures at
Trichrome Stains
which they can be seen as separate objects
 Allow greater distinctions among various
*0.2 micra is the maximal resolving power of the light
extracellular tissue components. (e.g. Masson
microscope
trichrome)
Virtual Microscopy
Periodic Acid-Schiff (PAS) Reaction
 Used for study of bright-field microscopic
 Utilizes the hexose rings of polysaccharides and
preparations, involves the conversion of a stained
other carbohydrate-rich tissue structures and
tissue preparation to high-resolution digital
stains such as macromolecules distinctly purple
images and permits study of tissues using a
or magenta.
computer or other digital device, without an actual
stained slide or a microscope.
Feulgen Reaction
 Modified PAS procedure that stains DNA.

Sudan Black
 Lipid-soluble dye

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  H-01 Histology and Its Methods of Study
Fluorescence Microscopy
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
 A phenomenon when certain cellular substances
are irradiated by light of a proper wavelength,
they emit light with a longer wavelength.
Fluorescent compounds
 Acridine orange – binds both DNA and RNA
 DAPI and Hoechst stains – bind DNA and are
used to stain cell nuclei, emitting a characteristic
blue fluorescence under UV.
Phase-Contrast Microscopy
Phase-Contrast Microscopy
 Uses a lens system that produces visible images
from transparent objects and, importantly, can be
used with living, cultured cells.
 Based on the principle that light changes speed
when passing through cellular and extracellular
structures with different refractive indices.
Differential Interference Microscopy
 A modification of phase-contrast microscopy with
Nomarski optics which produces an image of
living cells with a more apparent three-
dimensional aspect.
Confocal Microscopy
Confocal Microscopy
 Achieves high resolution and sharp focus by
using:
1. A small point of high-intensity light, often from
a laser
2. A plate with a pinhole aperture in front of the
image detector
 Include a computer-driven mirror system (the
beam splitter) to move the point of illumination
across the specimen automatically and rapidly.
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Polarizing Microscopy
Polarizing Microscopy
 Allows recognition of stained or unstained
structures made of highly organized subunits.
Birefringence
 Ability to rotate the direction of vibration of
polarized light
 A feature of crystalline substances or substances
containing highly oriented molecules such as
cellulose, collagen, microtubules and actin
filaments.
ELECTRON MICROSCOPY
Transmission Electron Microscopy
Transmission Electron Microscope
 An imaging system that permits resolution around
3nm and allows isolated particles magnified as
much as 400,000 times to be viewed in detail.
Heavy Metal Ions
 Added to fixatives or dehydrating agents used for
tissue preparation to improve contrast and
resolution in TEM which bind cellular
macromolecules, increasing their electron density
and visibility. (e.g. osmium tetroxide, lead citrate,
and uranyl compounds)
Cryofracture and Freeze Etching
 Techniques that allow TEM study of cells without
fixation or embedding and useful in the study of
membrane structure.
Scanning Electron Microscopy
Scanning Electron Microscopy
 Provides a high-resolution view of the surfaces of
cells, tissues and organs.
 Beam does not pass through the specimen
 Presents a three-dimensional view that appears
to be illuminated in the same waythat large
objects are seen with highlights and shadows
caused by light.
  H-01 Histology and Its Methods of Study
AUTORADIOGRAPHY
Autoradiography
 Method of localizing newly synthesized
macromolecules in cells or tissue sections.
Radioactively Labeled Metabolites
 Nucleotides, amino acids and sugars
 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.
CELL AND TISSUE CULTURE
Primary Cell Cultures
 Cells to be cultured are dispersed mechanically
or enzymatically from a tissue or organ and
placed with sterile procedures in a clear dish to
which they adhere, usually as a single layer.
 Study molecular changes that occur in cancer
 To analyze infectious viruses, mycoplasma and
some protozoa and for many routine genetic or
chromosomal analyses.
HeLa cells
 Cervical cancer cells form a patient identified as
Henrietta Lacks
 One of the first cell lines
Transformation
 Process that promotes cell immortality
ENZYME HISTOCHEMISTRY
Enzyme Histochemistry/Cytochemistry
 Method for localizing cellular structures using a
specific enzymatic activity present in those
structures.
Examples of enzymes:
 Phosphatases – remove phosphate groups from
macromolecules
 Dehydrogenases – transfer 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 – promotes the oxidation of
substances with the transfer of hydrogen ions to
hydrogen peroxide.
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Perls’ Prussian Blue
 Reaction for iron
 Used to diagnose the iron storage diseases,
hemochromatosis and hemosiderosis
PAS Amylase
 Reactions for polysaccharides
 To detect glycogenosis and
mucopolysaccharidosis
Alcian Blue
 Reactions for lipids and sphingolipids
 To detect sphingolipidosis
VISUALIZING SPECIFIC MOLECULES
A specific macromolecules present in a tissue section
may also be identified by using tagged compounds or
macromolecules that bind specifically with the molecule of
interest. Most commonly used labels are:
 Fluorescent compounds
 Radioactive atoms
 Peroxidase or other enzymes
 Metal (usually gold)
Examples of molecules that interact specifically with other
molecules include the following:
 Phalloidin – a compound extracted from
mushroom, Amanita phalloides, interacts strongly
with the actin protein of microfilaments.
 Protein A – purified from Staphylococcus aureus
bacteria, binds to the Fc region of antibody
molecules, and can therefore be used to localize
naturally occurring or applied antibodies bound to
cell structures.
 Lectins – glycoproteins derived mainly from plant
seeds, bind to carbohydrates with high affinity
and specificity.
Immunohistochemistry
Immunohistochemistry
 Based on specific reactions between an antigen
and antibodies labeled with visible markers, often
fluorescent compounds or peroxidase for light
microscopy and gold particles for TEM.
Direct Immunohistochemistry
 If the cell or tissue antigen of interest is detected
by directly binding a labeled primary antibody
specific for that antigen.
  H-01 Histology and Its Methods of Study

Indirect Immunohistochemistry
 Uses an unlabeled primary antibody that is
detected bound to its antigen with labeled
secondary antibodies.

Antigens Diagnosis
Specific cytokeratins Tumors of epithelial
origin
Protein and polypeptide Certain endocrine
hormones tumors
Carcinoembryonic antigen Glandular tumors, mainly
(CEA) of the digestive tract and
breast
Steroid hormone receptors Breast duct cell tumors
Antigens producedby Specific virus infections
viruses

Immunohistochemistry

Hybridization
 Specific binding between two single strands of
nucleic acid, which occurs under appropriate
conditions if the strands are complementary.

In Situ Hybridization
 Specific gene sequences or mRNAs of cells can
be detected microscopically using labelled
complementary DNA (cDNA) probes.

ENZYME HISTOCHEMISTRY

 Many steps in tissue processing, slide

preparation, and staining can introduce minor
artifacts such as spaces and precipitates that are
not normally present in the living tissue and must
be recognized.
 Sections of cells or tissues are essentially 2D
planes through 3D structures, and understanding
this fact is important for their correct interpretation
and study.