CHAPTER 1:
HISTOLOGY & ITS METHODS OF STUDY
Histology
-the study of the tissues of the body and how these tissues
are arranged to constitute organs
-the Greek root histo can be translated as either “tissue” or
“web,” both of which are appropriate because tissues are
usually webs of interwoven filaments and fibers, both
cellular and noncellular, with membranous linings.
Tissues
-have two interacting components:
1. Cells
 Produce the ECM and are also influenced
and sometimes controlled by matrix
molecules
 Cells and Matrix interact extensively, with
many components of the matrix recognized
by and attaching to cell surface receptors
 Cells and ECM form a continuum that
functions together
2. Extracellular Matrix (ECM)
 Consists of many kinds of macromolecules,
most of which form complex structures, such
as collagen fibrils and basement
membranes
 Supports the cells and the fluid that
transports nutrients to the cells, and carries
away their catabolites and secretory
products
I. PREPARATION OF TISSUES FOR STUDY
Most tissues studied histologically are prepared with this
sequence of steps (a):
1. Fixation
-Small pieces of tissue are placed in solutions of
chemicals that preserve by cross-linking proteins and
inactivating degradative enzyme
2. Dehydration
-The tissue is transferred through a series of
increasingly concentrated alcohol solutions, ending
in 100%, which removes all water
3. Clearing
-Alcohol is removed in toluene or other agents in
which both alcohol and paraffin are miscible
4. Infiltration
-The tissue is then placed in melted paraffin until it
becomes completely infiltrated with this substance
5. Embedding
-The paraffin-infiltrated tissue is placed in a small
mold with melted paraffin and allowed to be harden
6. Trimming
-the resulting paraffin block is trimmed to expose the
tissue for sectioning (slicing) on a microtome
Similar steps are used in preparing tissue for transmission
electron microscope (TEM), except special fixatives and
dehydrating solutions are used with smaller tissue samples
and embedding involves epoxy resins which become harder
than paraffin to allow every thin sectioning
(b) A microtome is used for sectioning paraffin-embedded
tissues for light microscopy. The trimmed tissue specimen is
mounted in the paraffin block holder. The paraffin sections are
placed in glass slides and allowed to adhere, deparaffinized,
and stained for light microscope study
Fixation
 To avoid tissue digestion by enzymes present
within the cells (autolysis) or bacteria and to preserve
cell and tissue structure, pieces of organs begin to
be treated as soon as possible after removal from the
body.
 The initial treatment – fixation – usually involves
immersion in solutions of stabilizing or cross-linking
compounds called fixatives
 One fixative used widely for light microscopy is
formalin, a buffered isotonic solution of 37%
formaldehyde
 Both formaldehyde and glutaraldehyde, a fixative
often used for electron microscopy, react with the
amine groups (NH2) of tissue proteins, preventing
their degradation. Glutaraldehyde reinforces this
fixing activity by being a dialdehyde capable of cross-
linking proteins
 A double-fixation procedure, using a buffered
glutaraldehyde solution followed by immersion in
buffered osmium tetroxide, is a standard method to
prepare tissue for such studies
 Osmium tetroxide preserves (and stains) membrane
lipids a well as proteins
Embedding & Sectioning
 Tissues are embedded in a solid medium facilitate
sectioning. In order to cut very thin sections, tissues
must be infiltrated after fixation with embedding
material that imparts a rigid consistency to the tissue
 Embedding materials include paraffin and plastic
resins
 Paraffin is used routinely for light microscopy,
resins for BOTH light and electron microscopy
 Paraffin embedding, or tissue impregnation, is
preceded by two other main steps: Dehydration and
Clearing
a) Dehydration,
 Water is extracted from the fixed
tissues by successive transfer
through a graded series of ethanol
and water mixtures, usually from
70% to 100% ethanol
 The ethanol is then replaced by an
organic solvent miscible with
BOTH alcohol and the embedding
medium
 As the solvent infiltrates the
tissues, they become more
transparent (undergo clearing)
b) Clearing
 The fully cleared tissue is then
placed in melted paraffin in an
oven at 52°C to 60°C. At such
temperatures the clearing solvent
 evaporates and the tissue is filled
with liquid paraffin
 Tissues to be embedded with plastic resin are also
dehydrated in ethanol and – depending on the kind of
resin used – subsequently infiltrated with plastic
solvents
 Plastic embedding avoids the higher temperatures
needed for paraffin embedding, which helps avoid
shrinkage and major distortion of tissue
 A hardened block containing tissue and paraffin is
placed in an instrument called microtome and sliced
by the steel blade into extremely thin sections
Staining
 Cell components such as nucleic acids with a net
negative charge (anionic) stain more readily with
basic dyes and are termed BASOPHILIC
 Cationic components, such as proteins with many
ionized amino groups, have affinity for acidic dyes
and are termed ACIDOPHILIC
 Examples of basic dyes are toluidine blue, alcian
blue, and methylene blue. Hematoxylin behaves
like a basic dye, staining basophilic tissue
components
 The main tissue components that ionize and react
with basic dyes do so because of acids in their
composition (DNA, RNA, and glycosaminoglycans)
 Acid dyes (eosin, orange G, and acid fuchsin) stain
the acidophilic components of tissues such as
mitochondria, secretory granules and collagen
 Of all staining methods, the simple combination of
hematoxylin and eosin (H&E) is used most
commonly
 Hematoxylin produces a dark blue or purple color,
staining DNA in the cell nucleus and other acidic
structure (such as RNA-rich portions of the
cytoplasm and the matrix of the cartilage)
 Other dyes such as the trichomes (Mallory stain,
Masson stain), are used in more complex histologic
procedures. The trichomes, besides showing the
nuclei and cytoplasm very well, help to distinguish
extracellular tissue components better than H&E
 DNA can be specifically identified and quantified in
nuclei using the Feulgen reaction, in which
deoxyribose sugars are hydrolyzed by mild
hydrochloric acid, followed by treatment with
periodic acid-Schiff (PAS) reagent.
 This PAS reaction is based on the transformation of
1,2 glycol groups present in the sugars into
aldehyde residues, which then react with Schiff
reagent to produce a purple or magenta color
 Many polysaccharides can also be demonstrated by
the PAS reaction. A very common free polysaccharide
in animal cells is glycogen
 Short branched chains of sugars (oligosaccharides)
are attached to specific amino acids of
glycoproteins, making most glycoproteins PAS-
positive
 Glycosaminoglycans (GAGs) are anionic,
unbranched long chain polysaccharides containing
animated sugars
 Many GAGs are synthesized while attached to a core
protein and are part of a class of macromolecules
called proteoglycans, which upon secretion make up
important parts of the ECM
 GAGs and many acidic glycoproteins DO NOT
undergo the PAS reaction
 Basophilic or PAS-positive material can be further
identified by enzyme digestion, pretreatment of a
tissue section with an enzyme that specifically digests
on substrate
 Counterstain is usually a single stain that is applied
separately to allow better recognition of nuclei and
other structures. In H&E staining, Eosin is the
counterstain to Hematoxylin
 Lipid-rich structures of cells are best revealed with
lipid-soluble dyes
 Frozen sections are stained in alcohol solutions
saturated with a lipophilic dye such as Sudan black,
which dissolves lipid-rich structures of cells
 Metal impregnation techniques usually using
solutions of silver salts are a common method of
visualizing certain ECM fibers and specific cellular
elements in nervous tissue
II. LIGHT MICROSCOPY
Bright-Field Microscopy
 The bright-field microscope includes an optical
system and mechanisms to move and focus the
specimen
 The optical components consist of three lenses
 Condenser
-collects and focuses a cone of light that
illuminates the object to be observed
 Objective Lens
-enlarges and projects the image of the object in
the direction of the eyepiece
 Eyepiece or Ocular Lens
-further magnifies the image and projects it onto
the viewer’s retina or a charge-coupled device
(CCD) highly sensitive to low light levels with a
monitor and camera
 The critical factor in obtaining crisp, detailed image
with a light microscope is its Resolving Power,
defined as the smallest distance between two
particles at which they can be seen as separate
objects. The maximal resolving power of the light
microscope is approximately 0.2 micrometers
Fluorescence Microscopy
 When certain cellular substances are irradiated by
light of a proper wavelength, they emit light with a
longer wavelength --- a phenomenon called
fluorescence
 In fluorescence microscopy, tissue sections are
usually irradiated with ultraviolet (UV) light and the
emission is in the visible portion of the spectrum
 The fluorescent substances appear brilliant on a dark
background
 Fluorescent compounds with affinity for specific cell
macromolecules may be used as a fluorescent stains.
(Acriding orange, which binds both RNA and DNA)
Phase-Contrast Microscopy
 Unstained cells and tissue sections, which are
usually transparent and colorless, can be studied with
these modified light microscopes
 Phase-contrast microscopy uses a lens system that
produces visible images from transparent objects
and, importantly, can be used with living, cultures
cells
 A modification of phase-contrast microscopy is
differential interference microscopy with Nomarski
objects, which produces an image of living cells with
a more apparent three-dimensional (3D) aspect

Confocal Microscopy
 Stray (excess) light reduces contrast within the image
and compromises the resolving power of the objective
lens. Confocal microscopy avoids these problems
and 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
 The point light source, the focal point of the lens,
and the detector’s pinpoint aperture are ALL
optically conjugated or aligned to each other in the
focal plane (confocal) and unfocused light does
NOT pass through the pinhole
 Confocal microscopes include a computer-driven
mirror system (the beam splitter) to move the point
of illumination across the specimen automatically and
rapidly
Polarizing Microscopy
 Allows the recognition of stained or unstained
structures made of highly organized subunits
 When normal light passes through a polarizing filter,
it exits vibrating in only one direction
 The ability to rotate the direction of vibration of
polarized light is called Birefringence and is a
feature of crystalline substances or substances
containing highly oriented molecules, such as
cellulose, collagen, microtubules and actin
filaments
III. ELECTRON MICROSCOPY
Transmission Electron Microscopy
 TEM is an imaging system that permits resolution
around 3 nm
 To improve contrast and resolution in TEM,
compounds with heavy metal ions are often added
to the fixative or dehydrating solutions used to
prepare the tissue. These include osmium
tetroxide, lead citrate and uranyl compounds,
which bind cellular macromolecules, increasing their
electron density and visibility
 TEM normally require very thin sections; therefore
tissue is embedded in a hard epoxy and sectioned
with a glass or diamond knife
 Cryofracture and Freeze Etching are techniques
that allow TEM study of cells without fixation or
embedding
 In cryofracture, very small tissue specimens are
rapidly frozen in liquid nitrogen and either fractured
or cut with a knife
Scanning Electron Microscopy
 SEM provides a high resolution view of the
surfaces of the cells, tissues, and organs
 Like TEM, this microscope produces and focuses a
very narrow beam of electrons, but in this
instrument the beam DOES NOT pass through the
specimen. Instead, the surface of the specimen is
first dried and spray-coated with a very thin layer of
heavy metal (often gold) through which electrons DO
NOT readily pass through
 SEM images are usually easy to interpret because
they represent 3D view that appears to be illuminated
from above
IV. AUTORADIOGRAPHY
 Microscopic autoradiography is a method id
localizing newly synthesized macromolecules
(DNA, RNA, protein, glycoproteins, and
polysaccharides) in cells or tissue sections
 Much information becomes available by
autoradiography of cells or tissues. If radioactive
precursor of DNA (such as tritium-labeled
thymidine) is used, it is possible to know which cells
in a tissue (and how many) are replicating DNA and
preparing to divide
 Autoaudiographs are tissue preparations in which
particles called silver grams indicate the cells or
regions of cells in which specific macromolecules
were synthesized just prior to fixation
V. CELL & TISSUE CULTURE
 In preparing cultures from a tissue or organ, cells
must be dispersed mechanically or enzymatically.
Once isolated, the cells can be cultivated in a clear
dish to which they adhere, usually as a single layer of
cells. Culture of cells isolated this way are called
Primary Cell Cultures
 Many cell types, once isolated from normal or
pathologic tissue, can be maintained in vitro for
long periods because they become immortalized
and constitute a permanent cell line
 Most cells obtained from normal tissues have a finite,
genetically programmed life span. Certain changes,
however, can promote cell immortality, a process
called transformation, and are similar to the initial
changes in a normal cell’s becoming a cancer cell
VI. ENZYME HISTOCHEMISTRY
 Enzyme histochemistry (cytochemistry) is a
method for localizing cellular structures using a
specific enzymatic activity present in those
structures
 Enzyme histochemistry involves the following:
(1) Tissue sections are immersed in a
solution containing the substrate of the
enzyme to be localized
(2) The enzyme is allowed to act on its
substrate
(3) At this stage or later, the section is 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 by
having color or electron density,
precipitates over the site of the
enzymes, allowing the region to be
localized microscopically
 Examples of enzymes that can be detected
histochemically include the following:
 Phosphatases
 -Split bond between a phosphate group and
phosphorylated molecules
-Both alkaline phosphatases (those with
maximum activity at an alkaline pH) and acid
phosphatases can be detected
 Dehydrogenases
-Remove hydrogen ions from one substrate and
transfer them to another
-Localized by incubating tissue sections in a
substrate solution containing a molecule that
receives hydrogen and precipitates as an
insoluble colored compound
-Mitochondria can be specifically identified by
this method, because dehydrogenases are
among the citric acid (Krebs) cycle enzymes of
this organelle
 Peroxidase
-Promotes the oxidation of substrate with the
transfer of hydrogen ions to hydrogen
peroxide, forming water molecules
-Cell or tissue sections are incubated in a
solution containing hydrogen peroxide and 3,3’-
diaminoazobenzidine (DAB), which is oxidized
in the presence of peroxidase to produce an
insoluble, brown, electron-dense precipitate
VII. VISUALIZING SPECIFIC MOLECULES
 Examples of molecules that interact specifically with
other molecules include the following:
 Phalloidin
-a compound extracted from a mushroom,
Amanita phalloides, and interacts strongly with
actin. Tagged with fluorescent dyes, phalloidin
is commonly used to demonstrate actin
filaments in cells
 Protein A
-obtained from Staphylococcus aureus bacteria
and binds to the Fc region of immunoglobulin
(antibody) molecules. Labeled protein A can
therefore be used to localize naturally
occurring or applied antibodies bound to cell
structures
 Lectins
-proteins or glycoproteins, derived mainly from
plant seeds, that bind to carbohydrates with
high affinity and specificity
-Fluorescently labeled lectins are used to stain
specific glycoproteins, proteoglycans, and
glycolipids and are used to characterize
membrane components with specific
sequences of sugar residues
Immunohistochemistry
 The body’s immune cells interact with and produce
antibodies against macromolecules – called
antigens – that are recognized as “foreign,” not a
normal part of the organism, and potentially
dangerous
 Antibodies belong to the immunoglobulin family of
glycoproteins, produced by lymphocytes
 Immunohistochemistry is very widely used to
detect specific proteins of interest cells and tissues.
This technique requires an antibody against the
protein that is to be detected, which means that the
protein must have been previously purified using
biochemical or molecular approaches so that
antibodies against it can be produced
 Polyclonal Antibodies are a mixture of antibodies
collected from the animal’s plasma, each capable of
binding a different region of protein x
 Different hybridoma clones produce different
antibodies against the several parts of protein x and
each clone can be isolated and cultures separately
so that the different antibodies against protein x can
be collected separately. Each of these antibodies is a
Monoclonal Antibody
 There are DIRECT and INDIRECT methods of
immunohistochemistry
a. Direct Method
-the antibody (either monoclonal or
polyclonal) is itself tagged with an
appropriate label
-uses an antibody made against the
tissue protein of interest and tagged
directly with a label such as
fluorescent compound or peroxidase
b. Indirect Method
-involves sequential application of two
antibodies and additional washing
steps
-the (primary) antibody specifically
binding protein x is NOT labeled
-the detectible tag is conjugated to a
secondary antibody
Hybridization Techniques
 Hybridization
-implies the specific binding between two
single strands of nucleic acid, which occurs
under appropriate conditions if the strands are
complementary
 Hybridization also occurs when nucleic acid
sequences in solution are applied directly to
prepared cells and tissue sections, a procedure
called in situ hybridization (ISH)
 The nucleotide sequences of interest are
detected with probes consisting of single-
stranded complementary DNA (cDNA)
VIII. INTERPRETATION OF STRUCTURES IN TISSUE
SECTIONS
Artifacts
-minor structural abnormalities not present in the
living tissue
 One such distortion is minor shrinkage of cells or
tissue regions produced by the fixative, by the
ethanol, or by the heat needed for paraffin
embedding
 Other artifacts may include small wrinkles in the
section and precipitates from the stain