Pediatric Consultant's Guide to Histology Methods

Maria Mirasol L. Tuaño, MD, DPPS
Pediatric Consultant

 HISTOLOGY = is the study of the tissues of
the body & how these tissues are
arranged to constitute organs.
◦ The Greek root “histo” = can be translated as
either “tissue” or “web” and both translations are
appropriate because MOST of the tissues are
webs of interwoven filaments & fibers, both
cellular & noncellular, with membranous linings.
◦ It involves all aspects of tissue biology, with the
focus on how cells’ structure & arrangement
optimize functions specific to each organ.

 Tissues are made of 2 interacting
components:
◦ Cells &
◦ Extracellular matrix

 EXTRACELLULAR MATRIX:
◦ Consists of many kinds of molecules, MOST
of w/c are highly organized & form complex
structures, such as collagen fibrils &
basement membranes.
◦ The main function once attributed to the
extracellular matrix were:
 to furnish mechanical support for the cells,
 To transport nutrients to the cells,
 & to carry away catabolites & secretory
products.

 We now know that, although the cells
produce the extracellular matrix, they are
also influenced & sometimes controlled by
molecules of the matrix.
 There is, thus, an intense interaction
between cells & matrix, w/ many
components of the matrix recognized by &
attaching to receptors present on cell
surfaces.

 Most of these receptors are molecules that
cross the cell membranes & connect to
structural components of the intracellular
cytoplasm.
 Thus, Cells & Extracellular matrix form a
continuum that functions together & reacts
to stimuli & inhibitors together.

 Each of the fundamental tissues is formed
by several types of cells & typically by
specific associations of cells & extracellular
matrix.
 These characteristic associations facilitate
the recognition of the many subtypes of
tissues by students.

 Most organs are formed by an orderly
combination of several tissues, except the
Central Nervous System, w/c is formed
almost solely by nervous tissue.
 The precise combination of these tissues
allows the functioning of each organ & of
the organism as a whole.

 The small size of cells & matrix components
makes histology dependent on the use of
microscopes.
 Advances in Chemistry, Molecular Biology,
Physiology, Immunology, & Pathology and
the interactions among these fields are
essential for a better knowledge of tissue
biology.

 Familiarity w/ the tools & methods of any
branch of science is essential for a proper
understanding of the subject.
 These chapters reviews several of the more
common methods used to study cells &
tissues & the principles involved in these
method

 Preparation of histological sections or
tissue slices - the MOST common procedure
used in the study of tissues that can be
studied w/ the aid of the light microscope.
 Under the Light Microscope, tissues are
examined via a light beam that is
transmitted through the tissue.

 Because tissues & organs are usually too
thick for light to pass through them, they
must be sectioned to obtain thin,
translucent sections, & then attached to
glass slides before they can be examined.

 The ideal microscope tissue preparation
should be preserved so that the tissue on
the slide has the same structure &
molecular composition as it had in the
body.

 However, as a practical matter this is
seldom feasible & artifacts, distortions, &
loss of components due to the preparation
process are almost always present.

 The Basic steps used in tissue preparation
for Histology are shown in Figure 1-1.

 Sectioning fixed &
Embedded Tissue.
 Most tissues studied histologically are
prepared as shown.
 (A) Small pieces of fresh tissue are
placed in Fixative solutions w/c
generally cross-link proteins,
inactivating degradative enzymes &
preserving cell structures.
 The fixed pieces then undergo
“Dehydration” by being transferred
through a series of increasingly more
concentrated alcohol solutions,
ending in 100% w/c effectively
removes all water from the tissue.
 The alcohol is then removed in a
Clearing solution miscible in both
alcohol & melted paraffin.

Embedded Tissue.
 When the tissue is then placed in
melted paraffin at 58°C it becomes
completely infiltrated w/ this
substance.
 All steps to this point are commonly
done today by robotic devices in
active histology or pathology
laboratories.
 After Infiltration the tissue is placed in
a small mold containing melted
paraffin, w/c is then allowed to
harden.
 The resulting paraffin block is
trimmed to expose the tissue for
sectioning (Slicing).

Embedded Tissue.
 Similar steps are used in preparing
tissue for transmission electron
microscope, except that smaller tissue
samples are fixed in special fixatives
& dehydration solutions are used that
are appropriate for Embedding in
epoxy resins w/c become much
harder than paraffin to allow very thin
sectioning.

 It is used for sectioning
paraffin-embedded tissues for
light microscopy.
 After mounting a trimmed
block w/ the tissue specimen,
rotating the drive wheel moves
the tissue-block holder up &
down.
 Each turn of the drive wheel
advances the specimen holder
a controlled distance, generally
between 1 & 10 µm, & after
each forward move the tissue
block passes over the steel
knife edge, w/c cuts the
sections at a thickness equal to
the distance the block
advanced.
 A Microtome

 Paraffin sections are then
adhered to glass slides,
deparaffinized, & stained for
microscopic examination.
 For transmission electron
microscopy sections < 1 µm
thick are prepared from resin-
embedded cells using an
ultramicrotome w/ a glass or
diamond knife.
 A Microtome

 If a permanent section is desired, tissues
must be fixed.
 To avoid tissue digestion by enzymes
present w/in the cells (Autolysis) or by
bacteria & to preserve the structure &
molecular composition, pieces of organs
should be promptly & adequately treated
before, or as soon as possible after,
removal from the animal’s body.

 This treatment –Fixation-can be done by
chemical or, les frequently physical
methods.
 In CHEMICAL FIXATION:
◦ The tissues are usually immersed in
solutions of stabilizing or cross-linking
agents called FIXATIVES.

◦ Because the fixatives needs some time to
fully diffuse into the tissues, the tissues
are usually cut into small fragments
before fixation to facilitate the penetration
of the fixative & to guarantee preservation
of the tissue.
◦ Intravascular perfusion of fixatives can be
used.

◦ Because the fixative in this case rapidly
reaches the tissues through the blood
vessels, fixation is greatly improved.

 FORMALIN – one of the best fixatives for
routine light microscopy.
◦ A buffered isotonic solution of 37%
formaldehyde.
 The chemistry of the process involved in
fixation is complex & NOT always
understood

 FORMALDEHYDE & GLUTARALDEHYDE :
◦ Another widely used fixative
◦ Are known to react w/ the amine groups
(NH2) of tissue proteins.
◦ In the case of Glutaraldehyde, the fixing
action is reinforced by virtue of its being a
dialdehyde, w/c can cross-link proteins.

 In view of the high resolution afforded by
the electron microscope, greater care in
fixation is necessary to preserve
ultrastructural detail.
 Toward that end, a double fixation
procedure, using a buffered glutaraldehyde
solution followed by a second fixation in
buffered osmium tetroxide, is a standard
procedure in preparations for fine structural
studies.

 The effect of osmium tetroxide is to
preserve & stain lipids & proteins.

 Tissue are usually embedded in a solid
medium to facilitate sectioning.
 To obtain thin sections w/ the microtome,
tissues must be infiltrated after fixation w/
embedding substances that impart a rigid
consistency to the tissue.

 Embedding materials include:
◦ Paraffin - used routinely for light
microscopy
◦ Plastic resins – used for both light &
electron microscopy

 The process of Embedding, or tissue
impregnation = is ordinarily preceded by 2
main steps:
◦ Dehydration &
◦ Clearing

 DEHYDRATION :
◦ The WATER is first extracted from the
fragments to be embedded by bathing
them successively in a graded series of
mixtures of ETHANOL & Water, usually
from 70% to 100% Ethanol (DEHYDRATION)

 CLEARING:
◦ The Ethanol is then replaced w/ a solvent
miscible w/ both Alcohol & the embedding
medium.
◦ As the tissues are infiltrated w/ this
solvent, they generally become
transparent (CLEARING).

 Once the tissue is impregnated w/ the
solvent, it is placed in melted paraffin in an
oven, typically at 52-60°C.
 The heat causes the solvent to evaporate, &
the spaces within the tissues become filled
w/ paraffin.
 The tissue together w/ its impregnating
paraffin hardens after removal from the
oven.

 Tissues to be embedded w/ Plastic resin are
also dehydrated in Ethanol & depending on
the kind of Resin used subsequently
infiltrated w/ plastic solvents.
 The Ethanol or the solvents are later
replaced by plastic solutions that are
hardened by means of cross-linking
polymerizers.

 Plastic embedding prevents the shrinking
effect of the high temperature needed for
paraffin embedding & gives little or no
distortion to the cells.
 The hard blocks containing the tissues are
then placed in an instrument called a
MICROTOME & are sliced by the
microtome’s steel or glass blade into
sections 1 to 10 µm thick.

 Remember that one micrometer (1 µm) =
1/1,000 of a millimeter (mm) = 10-6 m.
 Other units of distance commonly used in
histology are the:
◦ nanometer (1nm = 0.001µm = 10-6 mm
= 10-9 m) &
◦ angstrom (1 Å = 0.1 nm or 10-4 µm ).

 The sections are floated on water & then
transferred to glass to be stained.
 An alternate way to prepare tissue sections
is to submit the tissues to rapid freezing.
 In this process, the tissues are fixed by
freezing ( physical, NOT chemical fixation)
& at the same time become hard & thus
ready to be sectioned.

 A freezing microtome – the CRYOSTAT – is
then used to section the frozen block w/
tissue.
 Because this method allows the rapid
preparation of sections w/o going through
the long embedding procedure described
above, it is routinely used in hospitals to
study specimens during surgical
procedures.

 Freezing of tissues is also effective in the
histochemical study of very sensitive
enzymes or small molecules, since freezing,
unlike fixation, does NOT inactivate most
enzymes.
 Finally, because immersion in solvents such
as XYLENE dissolves cell lipids in fixed
tissues, frozen sections are also useful
when structures containing lipids are to be
studied.

 To be studied microscopically sections must
typically be stained or dyed because most
tissues are colorless.
 Methods of staining tissues have therefore
been devised that NOT only make the
various tissue components conspicuous but
also permit distinctions to be made
between them.

 The dyes stain tissue components more or
less selectively.
 Most of these dyes behave like acidic or
basic compounds & have a tendency to
form electrostatic (SALT) linkages w/
ionizable radicals of the tissues.
 Tissue components w/ a net negative
charge (ANIONIC) stain more readily w/
BASIC dye & are termed BASOPHILIC;

 Example of Basic dyes are:
◦ Toluidine blue
◦ Alcian blue &
◦ Methylene blue
◦ Hematoxylin – behaves like a basic dye,
that is, it stains the basophilic tissue
component.

 The main tissue component that ionize &
react w/ basic dyes do so because of acids
in their composition:
◦ Nucleic acids
◦ Glycosaminoglycans
◦ & acid glycoproteins

 Cationic components, such as proteins w/
many ionized amino groups, have affinity
for ACIDIC DYES & are termed ACIDOPHILIC.
 Examples of Acid dyes:
◦ Orange G
◦ Eosin &
◦ Acid fuchsin

 Cationic components, such as proteins w/ many
ionized amino groups, have affinity for ACIDIC
DYES & are termed ACIDOPHILIC.
 Examples of Acid dyes:
◦ stain the acidophilic component of tissues
such as :
 Mitochondria
 Secretory granules &
 Collagen

 The simple combination of HEMATOXYLIN &
EOSIN (H&E) = is used MOST commonly.
 Hematoxylin = stains DNA of the cell
nucleus & other acidic structures such as
RNA-rich portions of the cytoplasm & the
matrix of cartilage blue.
 Eosin = in contrast, stains other
cytoplasmic components & collagen pink.

Figure 1-2: (a) Micrograph stained
w/ Hematoxylin & Eosin
 With H&E, basophilic
cell nuclei are stained
purple while cytoplasm
stains pink.
Micrographs of the columnar epithelium
lining the small intestine

 Many other dyes, such as the TRICHOMES =
are used in different histologic procedures.
 Examples of trichomes:
◦ Mallory stain
◦ Masson stain
 The trichomes, besides showing the nuclei
& cytoplasm very well, help to distinguish
extracellular tissue components better than
H&E.

 A good technique for differentiating
Collagen is the use of PICROSIRIUS,
especially when associated w/ polarized
light.

 The chemical basis of other staining
procedure is more complicated than the
electrostatic interactions underlying
basophilia & acidophilia.
 DNA can be specifically identified &
quantified in nuclei using the FEULGEN
REACTION, in w/c deoxyribose sugars are
hydrolyzed by mild hydrochloric acid,
followed by treatment w/ PERIODIC ACID &
SCHIFF REAGENT (PAS).

 The PAS Technique (PERIODIC ACID &
SCHIFF REAGENT)
◦ Is based on the transformation of 1,2-
glycol groups present in the sugars into
aldehyde residues, w/c then react w/
Schiff reagent to produce a purple or
magenta color.
◦ Polysaccharides constitute an extremely
heterogenous group in tissues & occur
either in a free state or combined w/
proteins & lipids.

SCHIFF REAGENT) :
◦ Because of their hexose sugar content,
many polysaccharides can also be
demonstrated by the PAS in liver, striated
muscle, & other tissues where it
accumulates.

SCHIFF REAGENT) :
◦ Short branched chains of sugars
(oligosaccharides) are attached to specific
amino acids of Glycoproteins, making
most glycoproteins PAS-positive.
◦ Figure 1-2b shows an example of cells
stained by the PAS reaction.

Figure 1-2: (b) Micrograph
stained by Periodic acid –Schiff
(PAS) reaction for glycoproteins.
 With PAS, staining is most
intense at the cell surface, where
projecting microvilli have a
prominent layer of glycoproteins
(arrow head) & in the mucin-rich
secretory granules of goblet
cells.
 Cell surface glycoproteins &
mucin are PAS-positive due to
their high content of
oligosaccharides &
polysaccharides.
 The PAS-stained tissue was
counterstained w/ hematoxylin
to show the cell nuclei.
Micrographs of the columnar epithelium
lining the small intestine

SCHIFF REAGENT) :
◦ Glycosaminoglycans (GAGs) are anionic,
unbranched long-chain polysaccharides
containing aminated sugars.
◦ Many glycosaminoglycans are synthesized
while attached to a core protein &
constitute a class of macromolecules
called Proteoglycans, w/c upon secretion
make up important parts of the
extracellular matrix (ECM).

SCHIFF REAGENT) :
◦ Unlike a glycoprotein, a proteoglycan’s
carbohydrate chains are great in weight &
volume than the protein core of the molecule.
◦ GAGs & many acidic glycoproteins do NOT
undergo the PAS reaction, but because of
their high content of anionic carboxyl &
sulphate groups show a strong electrostatic
interaction w/ alcian blue & other basic stain.

SCHIFF REAGENT) :
◦ Basophilic or PAS-positive material can be
further identified by enzyme digestion
pretreatment of a tissue section w/ an
enzyme that specifically digests one
substrate, leaving other adjacent sections
untreated.

SCHIFF REAGENT) :
◦ For example, pretreatment w/
ribonuclease will greatly reduce
cytoplasmic basophilia w/ little effect on
chromosomes, indicating the importance
of RNA for the cytoplasmic staining.

SCHIFF REAGENT) :
◦ Similarly, free polysaccharides are
digested by amylase, w/c can therefore be
used to distinguish glycogen from
glycoproteins in PAS-positive material.

 In many staining procedures certain
structures such as nuclei become labelled,
but other parts of cells are often not visible.
 In this case a Counterstain is used to give
additional information.
 A Counterstain is usually a single stain that
is applied to a section by another method to
allow better recognition of nuclei or other
structures.

 Lipid-rich structures are best revealed w/
LIPID-SOLUBLE dyes to avoid the steps of
slide preparation that remove lipids such as
treatment w/ heat, xylene, or paraffin.
 Typically frozen sections are stained in
alcohol solutions saturated w/ a lipophilic
dye such as Sudan black.

 The stain dissolves in cellular lipid droplets
& other lipid-rich structures, w/c became
stained in black.
 Specialized methods for the localization of
cholesterol, Phospholipids, & glycolipids are
useful in diagnosis of Metabolic diseases in
w/c there are intracellular accumulations of
different kinds of lipids.

 In addition to tissue staining w/ dyes, Metal
impregnation techniques usually silver salts
are a common method of visualizing certain
ECM fibers & specific cellular elements in
nervous tissue.

 The whole procedure, from Fixation to
observing a tissue in a light microscope,
may take from 12 hours to 2 ½ days,
depending on the size of the tissue, the
fixative, the embedding medium, & the
method of staining.
 The final step before observation is
Mounting a protective glass coverslip on the
slide w/ adhesive mounting media.

 Conventional bright-field microscopy, as well
as Fluorescence, a phase-contrast,
differential interference, confocal, &
polarizing microscopy are all based on the
interaction of light & tissue components &
can be used to reveal & study tissue features.

 With the Bright-field microscope, widely
used by students, stained preparations are
examined by means of light that passes
through the specimen.
 The microscope is composed of mechanical
& optical parts (figure 1-3).
 The optical components consist of 3
systems of lenses.

Figure 1-3: Bright-Field Microscope
 Components & Light
path of a bright-field
microscope:
◦ Photograph of a bright-
field light microscope
showing its components
& the pathway of light
from the substage lamp
to the eye of the
observer.

 The optical system has 3
sets of lenses: a
condenser, a set of
objectives, & either one or
2 eyepieces.
 Condenser collects &
focuses light, producing a
cone of light that
illuminates the tissue
slide on the stage.

 Objective lenses enlarge &
project the illuminated image
of the object in the direction of
the eyepiece.
◦ For routine histological studies
objectives having 3 different
magnifications are generally
used :
◦ X4 for low magnification
observations of a large area
(field) of the tissue.
◦ X10 for medium
magnification of a smaller
field.
◦ X40 for high magnification of
more detailed areas.

 Eyepiece or ocular lens
further magnifies this image
another X10 & projects it
onto the viewer’s retina,
yielding a total
magnification of X40, X100,
or X400 (with permission,
from Nikon Instruments) ,
photographic film, or (to
obtain a digital image) a
detector such as a charge-
coupled device (CCD)
camera.

 The total magnification is
obtained by multiplying
the magnifying power of
the objective & ocular
lenses.

 The critical factor in obtaining a crisp,
detailed image w/ a light microscope is its
Resolving power.
 Resolving power = defined as the smallest
distance between 2 particles at w/c they
can be seen as separate objects.
◦ The maximal RP of the light microscope is
approximately 0.2 µm; this permits good
images magnified 1000-1500 times.

 Objects smaller or thinner than 0.2µm (such
as a ribosome, a membrane, or a filament
of actin) cannot be distinguished w/ this
instrument.
 Likewise, 2 objects such as Mitochondria
will be seen as only one object if they are
separated by less than 0.2 µm.

 The quality of the image-its clarity &
richness of detail depends on the
microscope’s resolving power.
 The magnification is of value only when
accompanied by high resolution.
 The resolving power of a microscope
depends mainly on the quality of its
objective lens.

 The eyepiece lens enlarges only the image
obtained by the objective; it does not
improve resolution.
 For this reason, when comparing objectives
of different magnifications, those that
provide higher resolving power .

 Fluorescence = When certain substances are
irradiated by light of a proper wavelength,
they emit light w/ a longer wavelength.
 In Fluorescence microscopy:
◦ Tissue sections are usually irradiated w/
ultraviolet (UV) light & the emission is in
the visible portion of the spectrum.
◦ The fluorescent substances appear
brilliant on a dark background.

 In Fluorescence microscopy:
◦ For this method, the microscope has a
strong UV light source & special filters
that select rays of different wavelengths
emitted by the substances.

Figure 1-4a.
 Fluorescent compounds
w/ affinity for specific
cell macromolecules may
be used as fluorescent
stains.
 Example: Acridine
orange, w/c binds both
DNA & RNA
 When observed in the
fluorescence microscope,
these nucleic acids emit
slightly different
fluorescence, allowing
them to be localized
separately in cells.

Figure 1-4a.
Components of cells in
culture are often stained
w/compounds visible by
fluorescence
microscopy.
 (a): Kidney cells stained
w/ acridine orange,
w/c binds nucleic acid.
Under a fluorescence
microscope, nuclear
DNA emits yellow light
& the RNA-rich
cytoplasm appears
reddish or orange.

Figure 1-4b.
 Other compounds such
as Hoechst stain &
DAPI specifically bind
DNA & are used to
stain cell nuclei,
emitting a
characteristic blue
fluorescence under UV.

Figure 1-4b.
 Another important
application of
fluorescence
microscopy is achieved
by coupling fluorescent
compounds to
molecules that will
specifically bind to
certain cellular
components & thus
allow the identification
of these structures
under the microscope.

Figure 1-4b.
 Antibodies labeled w/
fluorescent compounds
are extremely
important in
Immunohistological
staining.

Figure 1-4b.
 Components of cells in
culture are often
stained w/compounds
visible by fluorescence
microscopy.
 (b): The less dense
culture of kidney cells
stained w/ DAPI (4’,6-
diamino-2-
phenylindole) w/c
binds DNA, & w/
phalloidin, w/c binds
actin filaments.

Figure 1-4b.
 (b): The less dense
culture of kidney cells
 Nuclei of these cells
show a blue
fluorescence & actin
filaments appear
green.
 Important information
such as the greater
density of
microfilaments at the
cell periphery is readily
apparent.
 (Figure 1–4b, with
permission, from Drs. Claire E. Walczak and
Rania Risk, Indiana University School of
Medicine, Bloomington.)

 Some optical arrangements allow the
observation of unstained cells & tissue
sections.
 Unstained biological specimens are usually
transparent & difficult to view in detail,
because all parts of the specimen have almost
the same optical density.

Figure 1-5
 Phase-
contrast
microscopy,
however, uses a
lens system that
produces visible
images from
transparent
objects.

 Unstained cells’ appearance in 3 types of
Light microscopy.
 Neural crest cells growing as a single layer
in culture appear differently w/ various
techniques of light microscopy.

 These cells are unstained & the same field
of cells, including 2 differentiating pigment
cells, is shown in each photo.
◦ (a) Bright-field microscopy
◦ (b) Phase-contrast microscopy
◦ (c) Differential interference microscopy

Figure 1-5a
 (a): Bright-field
microscopy:
w/o fixation & staining,
only the 2 pigment cells
can be seen

Figure 1-5b
 (b): Phase-contrast
microscopy:
Cell boundaries, nuclei,
& cytoplasmic structures
w/ different refractive
indices affect in-phase
light differently &
produce an image of
these features in all the
cells.

Figure 1-5b
 (b): Phase-contrast
microscopy:
With or w/o differential
interference, is widely
used to observe live cells
grown in tissue culture.
All x200.
 (With permission, from Sherry
Rogers,
Department of Cell Biology and
Physiology, University of New
Mexico.)

Figure 1-5c
 (c): Differential
interference
microscopy:
Cellular details are
highlighted in a different
manner using Nomarski
optics.

 Is based on the principle that light changes
its speed when passing through cellular &
extracellular structures w/ different
refractive indices.
 These changes are used by the phase-
contrast system to cause the structures to
appear lighter or darker in relation to each
other.

 Because it does not require Fixation or
staining, Phase-contrast microscopy allows
observation of living cells & tissue cultures,
& such microscopes are prominent tools in
all cells culture labs.

 A related method of observing unstained
cells or tissue sections is the Nomarski
differential microscopy, w/c produces an
image w/ a more apparent three-
dimensional aspect than in routine phase-
contrast microscopy.

 With a regular bright-field microscope the
beam of light is relatively large & fills the
specimen.
 Stray light reduces contrast within the
image & compromises the resolving power
of the objective lens.

 It avoids stray light & achieves greater
resolution by using :
◦ (1) a small point of high-intensity light
provided by a laser &
◦ (2) a plate w/ a pinhole aperture in front
of the image detector.

 The point of light source, the focal point of
the lens, & the detector’s pinpoint aperture
are all optically conjugated or aligned to
each other in the focal plane (Confocal) &
unfocused light does not pass through the
pinhole.

 This greatly improves resolution of the
object in focus & allows the localization of
specimen components w/ much greater
precision than w/ the bright-field
microscope.

 Most Confocal microscopes include a
Computer-driven mirror system (the Beam
splitter) to move the point of illumination
across the specimen automatically &
rapidly.
 Digital images captured at many individual
spots in a very thin plane-of-focus are used
to produce an “Optical section” of that
plane.

 Moreover, creating optical sections at a
series of focal planes through the specimen
allows them to be digitally reconstructed
into a three-dimensional image.

 Although a very small spot of light
originating from one plane of the
section crosses the pinhole & reaches the
detector, rays originating from other planes
are blocked by the blind.
 Thus, only one very thin plane of the
specimen is focused at a time.

 The diagram shows the practical
arrangement of a confocal microscope.
 Light from a laser source hits the specimen
& is reflected.
 A beam splitter directs the reflected light to
a pinhole & a detector.

 Light from components of the specimen
that are above or below the focused plane is
blocked by the blind.
 The laser scans the specimen so that a
larger area of the specimen can be
observed.

 Allows the recognition of structures made
of highly organized molecules.
 When normal light passes through a
Polarizing filter (such as Polaroid), it exits
vibrating in only one direction.
 If a second filter is placed in the microscope
above the first one, w/ its main axis
perpendicular to the first filter, No light
passes through.

Figure 1-7.
 If, however, tissue
structures containing
oriented
macromolecules are
located between the
2 polarizing filters,
their repetitive
structure rotates the
axis of the light
emerging from the
polarizer & they
appear as bright
structures against a
dark background (Fig
1-7)

Figure 1-7.
 Birefringence = the
ability to rotate the
direction of vibration
of polarized light & is
a feature of
crystalline
substances or
substances
containing highly
oriented molecules,
such as cellulose,
collagen,
microtubules, &
microfilaments.

 Polarizing Light Microscopy:
◦ Produces an image only of material having
repetitive, periodic macromolecular
structure; features w/o such structure are
Not seen.

 Polarizing Light Microscopy:
◦ Shown here is a piece of thin mesentery
that was stained w/ red picrosirius, orcein,
& hematoxylin, & was then placed directly
on a slide & observed by bright-field &
polarizing microscopy.

Figure 1-7a
 A piece of thin
mesentery
 (a): Under routine
Bright-field
microscopy
Collagen fibers appear
red, along w/ thin dark
elastic fibers & cell
nuclei.

Figure 1-7b
 A piece of thin
mesentery
 (b): Under
Polarizing light
microscopy
Only Collagen fibers are
visible & these exhibit
intense birefringence &
appear bright red or
yellow; elastic fibers &
nuclei lack oriented
macromolecular
structure & are Not
visible.

 Transmission & scanning electron
microscopes are based on the interaction of
electrons & tissue components.
 The wavelength in the electron beam is
much shorter than of light, allowing a
thousand-fold increase in resolution.

Figure 1-8a
 (a) The TEM is an
imaging system
that permits
resolution around 3
mm.
 This high resolution
allows
magnification of up
to 400,000 times to
be viewed w/
details.

Figure 1-8a
 (a) The TEM :
 Unfortunately, this
level of magnification
applies only to
isolated molecules or
particles.
 Very thin tissue
sections can be
observed w/ details
at magnifications of
up to about 120,000
times.

Figure 1-8b
 (b): SEM :
 Permits pseudo-three-
dimensional views of
the surfaces of cells,
tissues, & organs.
 Like the TEM this
microscope produces &
focuses a very narrow
beam of electrons, but
in this instrument the
beam does Not pass
through the specimen.

Figure 1-8b
 (b): SEM :
 Instead the surface of
the specimen is first
dried & coated w/ a
very thin layer of
metal atoms through
w/c electrons do Not
pass readily.

Figure 1-8b
 (b): SEM :
 When the beam is
scanned from point
to point across the
specimen it interacts
w/ the metal atoms &
produces reflected
electrons or
secondary electrons
emitted from the
metal.

Figure 1-8b
 (b): SEM :
 These are captured
by a detector & the
resulting signal is
processed to produce
a black-and-white
image on a monitor.

Figure 1-8b
 (b): SEM :
 SEM images are usually
easily understood,
because they present a
view that appears to be
illuminated from
above, just our
ordinary macroscopic
world is filled w/
highlights & shadows
caused by illumination
from above.

 Is a method of localizing newly synthesized
macromolecules (DNA, RNA, proteins,
glycoproteins, & polysaccharides) in cells or
tissue sections.
 Radioactively labeled metabolites
(nucleotides, amino acids) incorporated into
the macromolecules emit weak radiation
that is restricted to the cellular regions
where the molecules are located.

 Radiolabeled cells or mounted tissue
sections are coated in a darkroom w/
photographic emulsion containing silver
bromide crystals, w/c act as microdetectors
of this radiation in the same way that they
respond to light in common photographic
film.

 After an adequate exposure time in
lightproof boxes the slides are developed
photographically.
 The silver bromide crystals reduced by the
radiation are reduced to small black grains
of metallic silver, indicating locations of
radiolabeled macromolecules in the tissue.

Figure 1-9
 This general
procedure can be
used in
preparations for
both Light
microscopy & TEM.

Figure 1-9
 Autoradiographs are
tissue preparations in
w/c particles called
Silver grains indicate
the regions of cells in
w/c specific
macromolecules were
synthesized just prior
to Fixation.

Figure 1-9
 Precursors such as
nucleotides, amino acids, or
sugars w/isotopes
substituted for specific
atoms are provided to the
tissues & after a period of
incorporation, tissues are
fixed, sectioned, & mounted
on slide or TEM grids as
usual.

Figure 1-9
 This processing removes all
radiolabeled precursors,
leaving only the isotope in
the fixed macromolecules.
 In a darkroom the slides are
coated w/ a thin layer of
chemicals like those in the
photographic film & dried.

Figure 1-9
 In a black box the isotope in
newly synthesized
macromolecules emits
radiation exposing the layer
of photographic chemicals
immediately adjacent to the
isotopes location.

Figure 1-9
 The minute regions of
exposed chemicals in the
photographic layer are
revealed as silver grains by
“developing” the preparation
as if it were film, followed by
microscopic examination .

Figure 1-9
 Shown here are autographs
from the salivary gland of a
mouse injected w/ ³H-
fucose 8 hr before tissue
fixation.
 Fucose is incorporated into
oligosaccharides & the
results reveal location of
newly synthesized
glycoproteins containing
such sugars.

Figure 1-9a
 (a): Black “silver grains”
are visible over regions
w/ secretory granules &
the duct indicating
glycoprotein locations.
X1500.

Figure 1-9b
 (b): The same tissue
prepared for TEM
autoradiography shows
silver grains w/ a coiled
or amorphous
appearance against
localized mainly over the
granules (G) & in the
gland lumen (L). X7500.
 (Figure 1–9b, with permission, from
Ticiano G. Lima and A. Antonio
Haddad, School of Medicine,
Ribeirão Preto,
 Brazil.)

 Live cells & tissues can be maintained &
studied outside the body.
 In a complex organism, tissues & organs
are formed by several kinds of cells.
 These cells are bathed in fluid derived from
blood plasma, w/c contains many different
molecules required for growth.

 Cell culture has been very helpful in
isolating the effects of single molecules on
specific type of cells.
 It also allows the direct observation of the
behavior of living cells under a phase
contrast microscope.
 Many experiments that cannot be
performed in the living animal can be
accomplished in vitro.

 The cells & tissues are grown in complex
solutions of known composition(salts,
amino acids, vitamins) to w/c serum
components or specific growth factors are
added.
 In preparing cultures from a tissue or
organ, cells must be initially dispersed
mechanically or enzymatically.

Figure 1-5
 Once isolated,
the cells can
be cultivated
in a clear dish
to w/c they
adhere,
usually as a
single layer of
cells (Figure
1-5).

 Culture of cells that are isolated in this way
are called Primary cell cultures.
 Many cell types once isolated from normal
or pathologic tissue have been maintained
in vitro ever since because they have been
immortalized & now constitute a permanent
cell line.

 Most cells obtained from normal tissues
have a finite, genetically programmed life
span.
 Certain changes, however (some related to
oncogenes), can promote cell immortality, a
process called Transformation, w/c are
similar to the initial changes in a normal
cell’s becoming a cancer cell.

 Because of improvements in culture
technology, most cell types can now be
maintained in the laboratory.
 All procedures w/ living cells & tissues must
be performed in a sterile area, using
solutions & equipment, to avoid
contamination w/ microorganism.

 As shown in the next chapter, Incubation of
living cells in vitro w/ a variety of new
fluorescent compounds that are
sequestered & metabolized in specific
compartments of the cell provides a new
approach to understanding these
compartments both structurally &
physiologically.

 Other histological techniques applied to
cultured cells have been particularly
important for understanding the locations &
functions of microtubules, microfilaments,
& other components of the cytoskeleton.

 Cell culture has been widely used for the
study of the metabolism of normal &
cancerous cells & for the development of
new drugs.
 This technique is also useful in the study of
parasites that grow only w/in cells, such as
Viruses, Mycoplasma, & some Protozoa.

 In Cytogenetic research, determination of
human karyotypes (the number &
morphology of an individual’s
chromosomes) is accumulated by short-
term cultivation of blood cells or fibroblasts
& by examining the chromosome during
Mitotic division.
 In addition, cell culture is central to
contemporary techniques of molecular
Biology & recombinant DNA technology.

 Indicates methods for localizing cellular
structures in tissue sections using the
unique enzymatic activity present in those
structures.
 To preserve these enzymes histochemical
procedures are usually applied to unfixed or
mildly fixed tissue, often sectioned on a
Cryostat to avoid adverse effects of Heat &
Paraffin on enzymatic activity.

 Enzyme histochemistry usually works in the
following way :
◦ (1) Tissue sections are immersed in a
solution that contains 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 w/ a marker compound;

following way :
◦ (4) This compound reacts w/ a molecule
produced by enzymatic action on the
substrate;

following way :
◦ (5) The final reaction product, w/c must
be insoluble & w/c is visible by Light or
Electron microscopy only if it is colored or
electron-dense, precipitates over the site
that contains the enzymes.
When examining such a section in the
microscope, one can see the Cell regions
(or Organelles) covered w/ a colored or
electron-dense material.

 Examples of enzymes that can be detected
histochemically include the following:
◦ Phosphatases split the bond between a
phosphate group & an alcohol residue of
phosphorylated molecules.
 The visible, insoluble reaction product of
phosphatases is usually Lead phosphate
or Lead sulfide.

Figure 1-10
◦ Phosphatases
◦ Both alkaline
phosphatases w/c
have their
maximum activity
at an alkaline pH
& acid
phosphatases can
be detected.

◦ Dehydrogenases remove hydrogen from
one substrate & transfer it to another.
 Like phosphatases, Dehydrogenases play
an important role in several hydrogen &
precipitates as an insoluble colored
compound.

◦ Dehydrogenases
 Mitochondria can be specifically
identified by this method, since
dehydrogenases are key enzymes in the
Citric acid (Krebs) Cycle of this organelle.

◦ Peroxidase, w/c is present in several types
of cells, promotes the oxidation of certain
substrates w/ the transfer of hydrogen
ions to hydrogen peroxide, forming
molecules of water.

◦ Peroxidase,
 In this method, sections of adequately
fixed tissue are incubating in a solution
containing hydrogen peroxide & 3,3’-
diamino-azobenzidine (DAB).

◦ Peroxidase,
 The latter compound is oxidized in the
presence of Peroxidase, resulting in an
insoluble, brown, electron-dense
precipitate that permits the localization
of Peroxidase activity by Light & electron
microscopy.

◦ Peroxidase,
 Peroxidase staining in White blood cells
is important in the diagnosis of certain
Leukemias.

Figure 1-10a
 (a): Micrograph of cross
sections of Kidney
tubules treated
histochemically by the
Gomori method for
Alkaline phosphatases
show strong activity of
this enzyme at the
apical surfaces of the
cells at the lumen of the
tubules.

Figure 1-10b
 (b): TEM image of a
Kidney cell in w/c acid
phosphatases has been
localized
histochemically in 3
lysosomes (Ly) near the
nucleus (N).
 The dark material w/in
these structures is Lead
phosphate that
precipitated in places
w/ acid phosphatase
activity.
 (Figure 1–10b, with permission, from
Eduardo Katchburian, Department of
Morphology, Federal University of Sao
Paulo, Brazil.)

 Many histochemical procedures are used
frequently in laboratory diagnosis, including:
◦ Perls’ Prussian blue reaction for Iron ( used
to detect the Iron storage diseases,
hemochromatosis &, Hemosiderosis),
◦ the PAS-amylase & Alcian blue reactions for
Glycogen & Glycosaminoglycans ( to detect
glycogenosis & Mucopolysaccharides), &
◦ reactions for lipids & sphingolipids (to
detect Sphingolipidosis)

Figure 1-11
 A specific
macromolecules
present in a tissue
section may sometimes
be identified by using
tagged compounds or
macromolecules that
specifically interact w/
the material of
interest.

 The compounds that will interact w/ the
molecule must be tagged w/ a label that
can be detected under the light or electron
microscope.

 The most commonly used labels are:
◦ fluorescent compounds - w/c can be seen
w/ a Fluorescence or laser microscope,
◦ radioactive atoms - w/c can be detected
w/ Autoradiography,
◦ Molecules of Peroxidase or other enzymes
– w/c can be detected w/ Histochemistry,
◦ & metal (usually GOLD) particles that can
be observed w/ Light & electron
microscopy.

 These methods can be used for detecting &
localizing specific sugars, proteins, &
nucleic acids.

 Labeling by Specific, High-affinity
interactions :
 Compounds or macromolecules that have
affinity toward certain cell or tissue
macromolecules can be tagged w/ a label &
used to identify that component &
determine its location in cells & tissues.

Figure 1-11 (1)
 Labeling by Specific,
High-affinity
interactions:
 (1) Molecule A has a
high & specific affinity
toward a portion of
molecule B.
◦ Examples: Antibody that
recognizes specific
antigens, usually
Proteins, or a segment of
single-stranded DNA w/
sequence-specific
complementarity to RNA
molecules in a cell.

Figure 1-11 (1)
High-affinity
interactions:
 (1) Molecule A can also
be a small compound
like Phalloidin, w/c
specifically binds actin
filaments, or a protein
as “protein A” w/c
binds all
Immunoglobulins.

Figure 1-11 (2)
High-affinity
interactions:
 (2) When A & B are
mixed, A binds to the
portion of B it
recognizes.

Figure 1-11 (3)
High-affinity
interactions:
 (3) Molecule A may be
tagged w/ a label that
can be visualized / a
light or electron
microscope.
◦ The label can be a
Fluorescent compound, a
enzyme such as
Peroxidase, an electron-
dense particle, or
radioisotope.

Figure 1-11 (4)
High-affinity
interactions:
 (4) If molecule B is
present in a cell or
extracellular matrix
that is incubated w/
labeled molecule A,
molecule B can be
detected & localized by
visualizing the labeled
molecule A & bound to
it.

 Example of molecules that interact
specifically w/ other molecules include the
following:
◦ Phalloidin – is a compound extracted from
the mushroom Amanita phalloides &
interacts strongly w/ actin.
 Tagged w/ Fluorescent dyes,
 Is commonly used to demonstrate actin
filaments in cells.

following:
◦ Protein A – is obtained from
Staphylococcus aureus & binds to the Fc of
immunoglobulin (Antibody) molecules.
 Labeled protein A can therefore be used
to localize naturally occurring or applied
antibodies bound to cell structures.

following:
◦ Lectins – are proteins or glycoproteins,
derived mainly from plant seeds & that
bind to Carbohydrates w/ high affinity &
specificity.
◦ Different Lectins binds to specific sugars
or sequence of sugar residues.

following:
◦ Lectins –
 Fluorescent labeled lectins are used to
stain specific Glycoproteins,
proteoglycans, & glycolipids and are
used to characterize membrane
components w/ specific sequence of
sugar residues.

 A highly specific interaction between
molecules is that between an antigen & its
antibody.
 For this reason, methods using labeled
antibodies have become extremely useful in
identifying & localizing many specific
proteins, Not just those w/ enzymatic
activity that can be demonstrated by
histochemistry.

 The body’s immune cells are able to
discriminate its own molecules (Self) from
foreign ones.
 When exposed to foreign molecules called
Antigens the body responds by producing
antibodies that react specifically & bind to
the antigen thus helping to eliminate the
foreign substance.

 Antibodies belong to the Immunoglobulin
family of glycoproteins, produced by
lymphocytes.
 In Immunohistochemistry, a tissue section
(or cells in culture) that one believes
contains the protein of interest is incubated
in a solution containing an antibody to this
protein.

 The antibody binds specifically to the
protein, whose location in the tissue or cell
can than be seen w/ either the light or
electron microscope, depending on the type
of compound used to label the antibody.

 Antibodies are commonly tagged w/
fluorescent compounds, w/ Peroxidase or
alkaline phosphatase for histochemical
detection, or w/ electron-dense gold
particles.

 For immunohistochemistry, one must have
an antibody against the protein that is to be
detected.
 This means that the protein must have been
previously purified using biochemical or
molecular approaches so that antibodies
against it can be produced.

 To produced antibodies against protein x of
a certain animal species (eg. A Human or
Rat ), the protein is first isolated & then
injected into an animal of another species
(eg. A rabbit or a goat ).
 If the protein’s amino acid sequence is
sufficiently different for this animal to
recognize it as foreign, that is, an antigen ,
the animal will produce antibodies against
the protein.

 Different groups (Clones) of lymphocytes in
the animal that was injected recognize
different parts of protein x and each clone
produces an antibody against that part.
 These antibodies are collected from the
animal’s plasma & constitute a mixture of
Polyclonal antibodies, each capable of
binding a different region of protein x.

 It is also possible, however, to inject protein
x into a mouse & then days later to isolate
the activated lymphocytes & place them into
culture.
 Growth & activity of these cells can be
prolonged indefinitely by fusing them w/
lymphocytic tumor cells to produce
Hybridoma cells.

 Different Hybridoma clones produce
different antibodies against the several
parts of protein x & each clone can be
isolated & cultured separately so that the
different antibodies against protein x can
be collected separately.
 Each of these antibodies is a Monoclonal
antibody.

 An advantage to using a monoclonal
antibody rather than polyclonal antibodies
is that it can be selected to be highly
specific & to bind strongly to the protein to
be detected, producing less nonspecific
binding to other proteins similar to the one
of interest.

 In the direct method of
immunocytochemistry, the antibody (either
monoclonal or polyclonal) is tagged itself
w/ an appropriate label.
 A tissue section is incubated w/ the
antibody for some time so that the antibody
interacts with & binds to protein x.

Figure 1-12
 The section is then
washed to remove
the unbound
antibody, processed
by the appropriate
method & examined
microscopically to
study the location
or other aspects of
protein x.

Figure 1-12
 Can be direct or
indirect.

Figure 1-12
 Direct
immunocytochemistry
- uses an antibody
made against the
tissue protein of
interest & tagged
directly w/ a label
such as a Fluorescent
compound or
Peroxidase.

Figure 1-12
 Direct
immunocytochemistry
-When placed w/ the
tissue section on a
slide, these labeled
antibodies bind
specifically to the
protein (Antigen)
against w/c they were
produced & can be
visualized by the
appropriate method.

Figure 1-12
 Indirect
immunocytochemistry
– uses 2 different
antibodies.
 A Primary antibody -
is made against the
protein (Antigen) of
interest & applied to
the tissue section first
to bind its specific
antigen.

Figure 1-12
 Indirect
immunocytochemistry
Then a labeled
Secondary antibody is
obtained that was :
(1) Made in another
vertebrae species
against
Immunoglobulin
proteins (antibodies)
from the species in w/c
the primary antibodies
were made & then

Figure 1-12
 Indirect
immunocytochemistry
Then a labeled
obtained that was :
(2) Labeled w/ a
Fluorescent compound
or Peroxidase.
When this labeled
secondary antibody is
applied to the tissue
section it specifically
binds the primary
antibodies, indirectly
labeling the protein of
interest on the slide.

Figure 1-12
 Indirect
immunocytochemistry
Then a labeled
obtained that was :
(2) Since more than one
labeled secondary
antibody can bind each
Primary antibody
molecule, labeling of the
protein of interest is
amplified by the direct
method.

 The Indirect method of
immunocytochemistry – is more sensitive
but requires 2 antibodies & additional
steps.
 Instead of labeling the (Primary) antibody
specific for protein, the detectable tag is
conjugated to a secondary antibody made
in a different “Foreign” species against the
immunoglobulin class to w/c the Primary
antibody belongs.

Figure 1-12
 Indirect
immunocytochemistry
detection – is
performed by initially
incubating a section of a
human tissue believed
to contain protein x w/
mouse anti-x antibody.
 After washing, the tissue
sections are incubated
w/ labeled rabbit or
goat antibody against
mouse antibodies.

Figure 1-12
 Indirect
immunocytochemistry
detection –
 This secondary
antibodies will
recognize the mouse
antibody that had
recognized protein x .
 Protein x can then be
detected by using a
microscopic technique
appropriate for the label
used for the secondary
antibody.

 The Indirect method of
immunocytochemistry :
 There are other indirect methods that
involve the use of other intermediate
molecules, such as the Biotin-avidin
technique.

Figure 1-13
 Examples of Indirect
immunocytochemistry,
demonstrating the use of
labeling methods w/ cells
in culture or after
sectioning for both light
microscopy & TEM.

Figure 1-13a
 Immunocytochemical
methods – to localize
specific proteins in cells can
be applied to either light
microscopic or TEM
preparations using a variety
of labels:
 (a) A decidual cell grown in
vitro stained to reveal a
mesh of intermediate
filaments throughout the
cytoplasm.

Figure 1-13a
methods :
 (a) Primary antibodies
against the protein Desmin,
w/c forms these
intermediate filament, &
FITC-labeled secondary
antibodies were used in an
indirect immunofluorescence
technique.
 The Nucleus is
counterstained light blue w/
DAPI.

Figure 1-13b
methods :
 (b): A section of small
intestine stained w/ an
antibody against the enzyme
lysozyme.
 The secondary antibody
labeled w/ Peroxidase was
then applied & the localized
brown color produced
histochemically w/ the
Peroxidase substrate DAB.

Figure 1-13b
methods :
 (b): A section of small
intestine
 The method demonstrates
lysozyme containing
structures in scattered
microphages & in the
clustered Paneth cells.
 Nuclei were counterstain w/
hematoxylin.

Figure 1-13c
methods :
 (c): A section of Pancreatic
acinar cells in a TEM
preparation incubated w/
antibody against the enzyme
Amylase antibody & then w/
protein A coupled w/ Gold
particles.
 Protein A has high affinity
toward antibody molecules &
the resulting image reveals
the presence of Amylase w/
the Gold particles

Figure 1-13c
methods :
acinar cells
 Protein A has high affinity
toward antibody molecules &
the resulting image reveals
the presence of Amylase w/
the Gold particles localized
as very small black dots over
dense secretory granules &
developing granules (left).

Figure 1-13c
methods :
acinar cells
 With specificity for
Immunoglobulin molecules,
labeled protein A can be
used to localize any Primary
antibody.
 (Figure 1–13c, with permission, from Moise
Bendayan, Departments of
Pathology and Cell Biology, University of
Montreal.)

 Immunocytochemistry has contributed
significantly to research in Cell biology & to
the improvement of medical diagnostic
procedures.
 Table 1-1 shows some of the routine
applications of Immunocytochemical
procedures in clinical practice.

Table 1-1: Many pathologic conditions are diagnosed by localizing
specific markers of the disorder using antibodies against those
antigens in immune-histochemical staining.
Antigens Diagnosis
Specific cytokeratins Tumors of epithelial origin
Protein & polypeptide hormones Protein or Polypeptide hormone-
producing endocrine tumors
Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the
digestive tract & breast
Steroid hormone receptors Breast duct cell tumors
Antigens produced by Viruses Specific virus infections

 The central challenge in modern cell biology
is to understand the workings of the cell in
molecular detail.
 This goal requires techniques that permit
analysis of the molecules involved in the
process of information flow from DNA to
Protein.
 Many techniques are based on Hybridization.

 Hybridization – is the binding between 2
single strands of nucleic acids (DNA w/
RNA, RNA w/ RNA, or RNA w/ DNA) that
recognize each other if the strands are
complementary.
◦ The greater the similarities of the
sequences, the more readily
complementary strands form “hybrid”
double-strand molecules.

 Hybridization thus allows the specific
identification of sequences of DNA or RNA.
 This is commonly performed w/ nucleic
acids in solution, but hybridization also
occurs when solution of nucleic acid are
applied directly to cells & tissue sections, a
procedure called in situ hybridization (ISH).

 This technique is ideal for :
◦ (1) determining if a cell has a specific
sequence of DNA (such as a Gene or part
of a gene),
◦ (2) identifying the cells containing specific
mRNAs ( in w/c the corresponding gene is
being transcribed), or
◦ (3) determining the localization of a gene
in a specific chromosome.

 DNA & RNA of the cells must be initially
denatured by Heat or other agents to
become completely single-stranded.
 They are then ready to be hybridized w/ a
segment of single-stranded DNA or RNA
(called a Probe) that is complementary to
the sequence one wishes to detect.

 The Probe may be obtained by cloning, by
PCR amplification of the target sequence, or
by chemical synthesis if the desired
sequence is short.
 The probe is tagged w/ nucleotides
containing a radioactive isotope (w/c can be
localized by autoradiography) or modified
w/ a small compound such as Digoxygenin
(w/c can be identified by
Immunocytochemistry).

Figure 1-14
 A solution containing
the probe is placed
over the specimen for
a period of time
necessary for
Hybridization.
 After washing off the
excess unbound probe,
the localization of the
hybridized probe is
revealed through its
label.

Figure 1-14
 In situ hybridization
shows that many of the
epithelial cells in this
section of a Genital
wart contain the
Human papillomavirus
(HPV), w/c causes this
benign proliferative
condition.

Figure 1-14
 In situ hybridization:
The section was
incubated w/ a
solution containing a
Digoxygenin-labeled
cDNA probe for the
HPV DNA.
 The probe was then
visualized by direct
immunohistochemistry
using Peroxidase-
labeled antibodies
against digoxgenin.

Figure 1-14
 In situ hybridization:
This procedure stains
brown only those cells
containing HPV. X400.
H&E counterstain.
 (With permission, from Jose E.
Levi, Virology Lab, Institute of
Tropical Medicine, University of
Sao Pãulo, Brazil.)

 A key point to be remembered in studying &
interpreting stained tissue sections is that :
◦ (1) Microscope preparations are the end
result of a series of processes that began
w/ collecting the tissue & ended w/
mounting a coverslip on the slide.
 Several steps of this procedure may distort
the tissues, producing minor structural
abnormalities called Artifacts.

 Structures seen microscopically then may
differ slightly from the structures present
when they were alive.
 One such distribution is minor shrinkage of
cells or tissue regions produced by the
Fixative, by the ethanol, or by the Heat
needed for Paraffin embedding.
 Shrinkage can produce the appearance of
artificial spaces between cells & other tissue
components.

 Another source of artificial spaces is the loss
of molecules such as Lipids, Glycogen, or low
molecular weight substances that are not
kept in he tissues by the Fixative or removed
by the dehydrating & clearing fluids.
 Slight cracks in sections also appear as large
spaces in the tissues.

 Other artifacts may include:
◦ Wrinkles of the section – w/c may be
confused w/ linear structures such as
Blood capillaries &
◦ Precipitates of stain – w/c may be
confused w/ cellular structures such as
Cytoplasmic granules.
 Students must be aware of the existence of
artifacts & able to recognize them.

◦ (2) Impossibility of differentiating staining
all tissue components on a slide stained
by a single procedure.
◦ With the Light microscope it is necessary
to examine several preparations stained
by different methods to obtain an idea of
the tissue’s complete composition &
structure.

 The TEM, on the other hand, allows the
observation of cells w/ all organelles &
inclusions, surrounded by the components
of the ECM.

◦ (3) Finally, when a three-dimensional
tissue volume is cut into very thin
sections, the sections appear
microscopically to have only 2
dimensions:
 Length &
 Width

 When examining a section under the
microscope, one must always keep in mind
that something may be missing in front of
or behind that section because many tissue
structures are thicker than the section.

Figure 1-15
 Round structures seen
microscopically may be
sections through
spheres or cylinders &
tubules in cross-
section look like rings.
 Also since structures
w/in a tissue have
different orientations,
their two-dimensional
appearance will vary
depending on the
plane of section.

Figure 1-15
 A single convoluted
tube will appear
histologically as
several rounded
structures.

Figure 1-15a
 3-D structures appear
to have only 2-D in
thin sections:
 (a) Sections through a
hollow swelling on a
tube produce large &
small circles, oblique
sections through bent
regions of the tube
produce ovals of
various dimensions.

Figure 1-15b
to have only 2-D in
thin sections:
 (b) A single section
through a highly coiled
tube shows many
small, separate round
or oval sections.

Figure 1-15b
to have only 2-D in
thin sections:
 (b) On first observation
it may be difficult to
realize that these
represent a coiled
tube, but it is
important to develop
such interpretive skill
in understanding
histological
preparations.

Figure 1-15c
to have only 2-D in
thin sections:
 (c) Round structures in
sections may be
portions of either
spheres or cylinders.
 Additional sections or
the appearance of
similar nearby
structures help reveal a
more complete picture.

 To understand the architecture of an organ,
one often must study sections made in
different planes.
 Examining many parallel sections (Serial
sections) & reconstructing the images 3-
dimensionally provides better
understanding of a complex organ or
organism.

Pediatric Consultant's Guide to Histology Methods

Pediatric Consultant's Guide to Histology Methods

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