CHAPTER 22: IMMUNOHISTOCHEMISTRY (IHC) POLYCLONAL AND MONOCLONAL ANTIBODIES

INTRODUCTION Animals immunized with the specific immunogen will produce numerous clones of plasma cells (polyclonal)
that in turn will produce the antibody
Immunohistochemical Techniques are:
POLYCLONAL ANTIBODIES MONOCLONAL ANTIBODIES
→ routinely used for the identification of specific or highly selective cellular epitopes or antigens in frozen or • produced by immunizing an animal with a • Monoclonal antibodies are the products of an
paraffin-embedded tissues purified specific molecule (immunogen) individual clone of plasma cells
→ used to detect organisms in cytologic preparations such as: that contains the antigen of interest and • Hybridoma and Cloning Techniques have
✓ fluids collecting immunoglobulin-rich serum after the been developed to produce monoclonal
✓ sputum samples animal has produced humoral antibody antibodies that do not cross-react with other
✓ fine needle aspirates against the antigen molecules
• polyclonal antibodies are produced by • Antibodies from a given clone are
Immunologic Techniques make use of antigen-antibody interactions, whereby the site of antigen binding different cells of the animal – they are immunochemically identical and react with a
is demonstrated by: immunochemically not identical to each other specific epitope on the antigen against which
(1) DIRECT LABELING of the antibody thus reacting with various epitopes on the they are raised
(2) SECONDARY LABELING method antigen against which they are raised o Mice are currently used almost
• Some of the polyclonal antibodies may cross- exclusively for the production of
ANTIBODY belong to the class of serum proteins known as “immunoglobulins” react with other molecules and cause non- monoclonal antibodies
IgG the most commonly used antibody for immunocytochemistry specific staining, requiring: • Propagation can be carried out in culture
EPITOPE the structural part of the antigen that reacts with an antibody ► purification by absorption with the medium or by Transplantation of the
appropriate antigen hybridoma into the peritoneal cavity of
Immunohistochemistry (IHC) ► antibody dilution to eliminate the syngeneic mice from where the antibodies are
unwanted reaction harvested
• The most frequently used animal for the o This has dramatically increased the
• combines anatomical, immunological and biochemical techniques to identify discrete tissue
production of polyclonal antibodies is the quantities and number of specific
components by the interaction of target antigens with specific antibodies tagged with a visible label
rabbit, followed by: monoclonal antibodies available for
✓ goat, pig, sheep, horse, guinea pig and immunohistochemistry
• visualization of the distribution and localization of specific cellular components within cells
others
• used for disease diagnosis and biological research
TISSUE PREPARATION FOR IMMUNOHISTOCHEMISTRY
Example: Specific tumor markers use IHC to diagnose a cancer as benign or malignant, determine the
• Tissue must be prepared as a cryostat section and fixed in absolute methanol or acetone to:
stage and grade of a tumor, and identify the cell type and origin of metastasis to find the site of the
✓ preserve immunological activity
primary tumor
✓ prevent destruction of some of the labile antigenic sites
• also used for drug development to test drug efficacy by detecting either the activity or the up- or down-
• Immunofluorescence and Immuno-peroxidase techniques may also be done on formaldehyde-fixed
refulation of disease targets
and paraffin embedded sections
Immunofluorescence is often, but not always performed on frozen tissue due to the high background
auto-fluorescence seen in formalin fixed paraffin embedded tissue • Many masked antigens can now be retrieved in routinely processed tissue by:
(1) Proteolytic Enzyme Digestion
All of the steps involved in IHC Methodology are separated into two groups: (2) Microwave Antigen Retrieval
(3) Microwave and Trypsin Antigen Retrieval
(4) Pressure Cooker Antigen Retrieval
(1) SAMPLE PREPARATION

− samples are prepared on individual slides or multiple samples can be arranged on a single
slide for comparative analysis, such as with tissue microarrays
− samples can be viewed by either LIGHT MICROSCOPY or FLUORESCENCE MICROSCOPY

(2) LABELING
 PROTEOLYTIC ENZYME DIGESTION PRE-TREATMENT OF TISSUE SECTIONS

• Formalin fixed paraffin sections are usually pre-treated with proteolytic enzymes to: Antigenic determinants masked by formalin-fixation and paraffin-embedding often may be exposed by epitope
✓ break down formalin cross-linking unmasking, enzymatic digestion or saponin, etc.
✓ unmask and allow certain antigenic sites to be exposed → Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded
• Proteolytic enzyme digestion is especially useful for:
✓ demonstrating heavy chain immunoglobulins Procedure:
✓ complement and specific antigens (i.e. cytokeratin) in formalin-fixed paraffin-embedded biopsies
1. Rinse sections in PBS-Tween 2 times for 2 minutes each time.
The most common enzymes used are trypsin and protease. 2. Serum Blocking: Incubate sections with normal serum block – species same as secondary antibody,
for 30 minutes to block non-specific binding of immunoglobulin.
Before pre-treatments are employed, the sections are: Note: This protocol uses avidin-biotin detection system. Avidin-biotin block may be needed based on
tissue type. Normal serum block should be used prior to avidin-biotin block.
3. Primary Antibody: Incubate sections with primary antibody at appropriate dilution in primary
1. Deparaffinized
antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
4. Rinse in PBS-Tween 20.
2. Taken to alcohol
5. Peroxidase Blocking: Incubate sections in peroxidase blocking solution for 10 minutes at room
temperature.
Peroxidase Labeling
6. Rinse in PBS-Tween 20.
7. Secondary Antibody: Incubate sections with biotinylated secondary antibody at appropriate dilution
treated with 0.5% methanol in hydrogen peroxide for 10-15 minutes
in PBS for 30 minutes at room temperature.
(to destroy endogenous peroxidase activity)
8. Rinse in PBS-Tween 20 3 times for 2 minutes each time.
9. Detection: Incubate sections in streptavidin-HRP in PBS for 30 minutes at room temperature.
3. The slides are then washed in running water and taken to distilled water. 10. Rinse in TBS 3 times for 2 minutes each time.
11. Chromogen/Substrate: Incubate sections in DAB solution for 1-3 minutes.
Trypsin Method 12. Rinse in PBS-Tween 20 2 times for 2 minutes each time.
13. Counterstain if desire.
0.1% trypsin in 0.1 % calcium chloride in distilled water, adjusted to pH 7 .8 with sodium hydroxide 14. Rinse in distilled water.
15. Dehydrate through 95% ethanol for 2 minutes, then 100% ethanol for 2 times 3 minutes each time.
The slides are preheated at 37°C in distilled water before placing in freshly prepared trypsin 16. Clear in xylene.
solution 17. Coverslip with mounting medium.

4. The slide is transferred to cold running water to terminate enzyme digestion. HEAT-INDUCED EPITOPE RETRIEVAL (HIER)

Protease Method
• a simple and essential part of many immunohistochemistry and in situ hybridization procedures, is a
pretreatment method used to improve staining results
0.05 to 0.1% protease in distilled water, adjusted to pH 7.8 with sodium hydroxide
Heat coupled with specific buffered solutions
The section is preheated at 37°C in distilled water and placed in protease solution for a shorter
period of time due to its faster rate of enzyme digestion.
→ used to recover antigen reactivity in formalin fixed paraffin embedded tissue
→ it reverses the formaldehyde mediated chemical modifications of the antigen; thermal energy:
PARAFFIN SECTIONS ► breaks the crosslinks that bind surrounding proteins or peptides to the antigen which lead
to the “opening” or “unmasking” the epitope
1. Deparaffinize sections in xylene 2 times for 5 minutes each time. ► removes bound calcium ions from the sites of cross-links since several HIER buffers,
2. Hydrate with 100% ethanol 2 times for 3 minutes each time. (EDTA and citrate) act as calcium chelators
3. Hydrate with 95% ethanol for 1 minute.
4. Rinse in distilled water.
5. Follow procedure for pretreatment as required. HIER heating sources include the microwave, vegetable steamer, pressure cooker and water bath
 → Microwave distribution of heat within a microwave is uneven or inconsistent which results in a lack
of reproducibility as to staining intensities
→ Pressure cooker, steamer and water bath produce uniform and consistent heat distribution
• In general, the higher the temperature of the HIER solutions, the more effective the recovery of
the epitope is.
o the higher temperatures produced by the pressure cooker are advantageous however, damage or
distortion to the morphology of connective tissues can also occur
Reagents Required for Heat-induced Epitope Retrieval:
✓ 10 x Antigen Retrieval Solution
» Antigen Retrieval Reagent-Basic
» Antigen Retrieval Reagent-Acidic
» Antigen Retrieval Reagent-Universal
✓ Deionized H2O
✓ 1 x PBS: 0.137 M NaCl, 0.05 M NaH2PO4, ph 7.4
Equipment:
✓ Polypropylene Coplin staining jar (or equivalent)
✓ Water bath at 92-95oC
Procedure:
1. Make working dilutions by mixing 1 part of 10 x Antigen Retrieval concentrate with 9 parts of
deionized water.
2. Preheat retrieval solution to 92-95°C. This may be achieved by placing a polypropylene Coplin
staining jar filled with retrieval solution into a water bath.
Note: Heating may cause cracking of glass staining dishes.
3. Immerse slides into preheated retrieval solution for 2-10 minutes. Note: Since the effect of antigen
retrieval reagents depends on their temperature (90-100 °C) and incubation time (up to 30
minutes), optimal conditions should be determined by the individual investigator. Cryostat sections
are more sensitive to damage by retrieval solution than paraffin-embedded tissues. To avoid tissue
damage, it may be necessary to shorten the incubation time to 2-5 minutes.
4. After the incubation is finished, remove the Coplin jar with retrieval solution and slides from the
water bath, and let it cool to room temperature.
5. Gently rinse the slides with deionized water and then with PBS.
Note: Because tissues may be loosened after the retrieval procedure, avoid vigorous rinsing to
prevent detachment from the slides.
• Antibodies are demonstrated well on paraffin sections after heat pre-treatment such as the:
✓ proliferation markers (Ki-67 and MIB-1)
✓ hormone receptors (ER and PR)
✓ growth factor receptors (HER-2/neu)
✓ and others which were previously thought to be applicable only to frozen sections,.
• Most antigen retrieval methods apply temperatures near the boiling point of water.
• The optimal length of exposure to heat may vary from 10-60 minutes and depends to some extent on the
length of formalin fixation.
o 20 minutes is the most satisfactory time period for most antigens and fixation protocols.
Care should be taken not to allow the sections to dry after heating, as this destroys antigenicity
• Boiling of poorly fixed material often damages nuclear details, fibrous and fatty tissues tend to detach
from the slide. This can be prevented by:
► mounting the sections on slides with a strong adhesive (such as Vectabond)
► dipping Vectabond-coated slides in I0% formol saline for 1-2 minutes
► air drying before picking up sections
→ Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction
(PCR) is increasingly being used to detect viral genomes and oncogene mutations.
→ On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72-hour
of fixation time.
→ On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix,
and after 48 hours in the formalin-fixed tissue.
→ Bouin's and B-5 tissues give consistent results only after 6 hours of fixation
The choice of fixative and fixation time are critical factors influencing the outcome of PCR
amplification of nucleic acids from paraffin-embedded material.
In the large batch microwave oven technique, heating temperature is not uniformly distributed and
slides are subjected to "hot spots" and "cold spots" resulting in inconsistent antigen recovery.
PRESSURE COOKER ANTIGEN RETRIEVAL
• An alternative that appears to be less time consuming and allows for more consistent recovery of
many antigens, compared to large batch microwave oven technique.

MICROWAVE ANTIGEN RETRIEVAL ANTIGENS

• A relatively new technique that involves the boiling of formalin-fixed deparaffinized sections in certain Primary antibodies against numerous antigens are now available in the market, and are widely used for
solutions, such as: diagnosis of tumors, determination of tumor type, the evaluation of prolife-ration potential, identification of
✓ 0.01 M-citrate buffer (pH 6.0) infectious agents, prognostic and therapeutic implications, and many other aspects of diagnostic pathology.
✓ EDTA at pH 8.0
✓ Tris EDTA (pH or 10.0)

• Many antigens thought previously to be either lost or destroyed by routine histological processing
techniques can be retrieved by microwave oven heating
 MARKERS 3) CARCINOEMBRYONIC ANTIGEN (CEA)

EPITHELIAL TUMOR MARKERS − an oncofetal antigen especially useful for differentiating between adenocarcinoma (CEA-positive)
and mesothelioma (CEA-negative)
1) KERATIN
POSITIVE NON-REACTIVE NEGATIVE
− a highly sensitive marker for epithelial cells and present in epithelial tumors (carcinoma) Carcinomas of the: ✓ Prostate carcinoma ✓ Mesothelioma
− certain non-epithelial tumors (mesotheliomas and non-seminomatous germ cell tumors) also stain ✓ gastrointestinal tract ✓ Thyroid carcinoma
positive for keratin ✓ pancreas ✓ Renal carcinoma
» may be distinguished from carcinoma by applying an additional panel of antibodies ✓ lung
✓ breast
more frequently found in carcinomas of the ✓ ovary
lung, breast, uterus and ovaries (serous tumors) ✓ uterus
CK7 (Cytokeratin 7)
✓ cervix
These tumors are typically negative (-) for CK20
is more common in carcinomas of the colon and stomach 4) THYROID TRANSCRIPTION FRACTOR-1 (TTF-1)
CK20 (Cytokeratin 20)
These tumors are usually negative (-) for CK7. − useful in distinguishing lung adenocarcinomas from mesotheliomas

CK7 and CK20 POSITIVE

POSITIVE NEGATIVE
✓ Transitional cell carcinomas of the bladder ✓ Renal cell carcinomas
✓ Mucinous ovarian tumors ✓ Hepatocellular carcinomas
✓ Prostatic adenocarcinomas
✓ Thyroid carcinomas
✓ Squamous cell carcinomas
(skin, lung and esophagus)
2) EPITHELIAL MEMBRANE ANITGEN (EMA)
✓ Thyroid (medullary thyroid carcinomas)
✓ Lung (small cell tumors of the lung)
✓ Neuroendocrine tumors
✓ Carcinoid tumors
5) PROSTATE SPECIFC ANTIGEN
− extremely useful in the diagnosis of Prostatic Adenocarcinoma
− it is also positive in certain pancreatic and salivary gland tumors

− a high molecular weight protein helpful in determining the site of tumor INTERMEDIATE FILAMENT MARKERS

POSITIVE NON-REACTIVE NEGATIVE 1) ACTIN

Adenocarcinomas ✓ Hepatocellular Non-epithelial tumors
of the: carcinomas ✓ Sarcomas − is a contractile intermediate filament protein present in muscle and some non-muscle tissue
✓ breast ✓ Adrenal carcinomas ✓ Lymphomas − it is a sensitive marker for muscle differentiation
✓ lung ✓ Embryonal carcinomas ✓ Melanomas − can be used to identify tumors derived from smooth, skeletal and cardiac muscle.
✓ kidneys Other tumors
✓ Meningiomas 2) VIMENTIN
✓ Mesotheliomas
✓ Anaplastic large cell lymphomas − is a 57kD intermediate filament that is present in normal mesenchymal cells and their neoplastic
✓ Plasma cell tumors counterparts (i.e., sarcoma, melanoma, lymphoma, leukemia, seminoma, and some neural tumors).
− Melanomas and Schwannomas always stain positive for vimentin, so that a negative staining
may be used to exclude the diagnosis
− it is almost always present in tissue sections because of the background stromal elements, and has
limited use as a stand-alone stain, but it can be very helpful when combined with other specific
tumor markers
3) DESMIN 2) CHROMOGRANIN

− is a 53 kD intermediate filament expressed by smooth and striated (skeletal and cardiac) muscle − is found in the neural secretory granules of endocrine tissues, and is recognized as a marker for
− it is considered to be highly specific for myogenic tumors, including: neuroendocrine differentiation.
► leiomyoma (smooth muscle tumor) − Immunoreactivity is typically granular and its distribution is similar to that seen with silver staining
► rhabdomyosarcoma (skeletal muscle tumor) methods such as Grimelius stain.
− it is also used to demonstrate the myogenic component of mixed tumors (i.e., carcinosarcomas or
malignant mixed mesodermal tumors) CHROMOGRANIN, KERATIN (+) CHROMOGRANIN (+) KERATIN (-)
neuroendocrine carcinoma paraganglioma
4) GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP)
3) SYNAPTOPHYSIN
− is a 51 kD intermediate filament protein expressed by central nervous system glial cells,
particularly astrocytes. − is a 38 kD transmembrane protein associated with presynaptic vesicles of neurons
− it is most widely used to confirm the diagnosis of astrocytoma (but may also be present in − it has been identified in normal neurons and neuroendocrine cells
certain cases of ependymomas, oligodendrogliomas and medulloblastomas).
− Non-CNS tumors (meningiomas, metastatic carcinomas and lymphomas) stain negative for GFAP. GERM CELL TUMOR MARKERS

5) NEUROFILAMENT (NF) Non-seminomatous germ cell tumors (i.e. embryonal carcinomas, teratomas, choriocarcinomas, and
endodermal sinus or yolk sac tumors) generally stain positive for epithelial markers (keratin). For more specific
− is expressed in cells of neural origin, particularly: classification, the following germ cell tumor markers are used:
✓ neurons
✓ neuronal processes 1) HUMAN CHORIONIC GONADOTROPIN (HCG)
✓ peripheral nerves
✓ sympathetic ganglia − is synthesized by placental syncytiotrophoblasts, and is a marker for choriocarcinoma
✓ adrenal medulla
✓ neuroendocrine cells 2) ALPHA-FETOPROTEIN (AFP)
− tumors that show neuronal or neuroendocrine differentiation (e.g., neuroblastomas,
ganglioneuromas, neuromas, chemodectomas, and pheochromocytomas) will stain positive for − is synthesized by normal liver hepatocytes, and is used as a marker for endodermal sinus
neurofilament tumors showing yolk sac differentiation

6) S-100 PROTEIN POSITIVE

✓ Embryonal carcinomas and teratomas
− is a low molecular weight calcium-binding protein that is expressed in: ✓ Hepatocellular carcinomas
✓ CNS glial cells
✓ Schwann cells 3) PLACENTA-LIKE ALKALINE PHOSPHATASE (PLAP)
✓ Melanocytes
✓ Histiocytes − is produced by the placental syncytiotrophoblasts in late pregnancy, and is used as a marker for
✓ Chondrocytes germ cell tumors, particularly germinomas
✓ skeletal and cardiac muscle
✓ myoepithelial cells POSITIVE
✓ some epithelial cells of breast, salivary and sweat gland epithelium ✓ Embryonal carcinomas
✓ Choriocarcinomas
NEUROENDOCRINE MARKERS ✓ Endo-dermal sinus tumors
PLAP is positive in majority of seminomas
1) NEURON-SPECIFIC ENOLASE (NSE)

− is an isoenzyme marker whose presence in tissue provides strong evidence of neural or

neuroendocrine differentiation.
 MESENCHYMAL TUMOR MARKERS Cancer-associated genes

1) MYEGENIC TUMORS The development and progression of a malignant phenotype of human tumors is related to abnormalities of
structure or activity of proto-oncogenes and/or mutation of tumor suppressor genes such as p53. Many
− Tumors of skeletal muscle origin are positive for muscle-specific actin and desmin and/or other cellular oncogenes, including c-erbB-2, c-myc and ras have been found to be activated in cancer,
muscle markers such as: particularly of the breast.
► myo-D1
► myoglobin INFECTIOUS AGENT MARKERS
► myogenin
Antigenic markers are now available for a number of infectious agents, including:
2) FIBROHISTIOCYTIC TUMORS ✓ Hepatitis A virus
✓ Hepatitis B surface and core antigens
− the use of histiocytic markers such as CD68, or FAM 56, combined with more non-specific ✓ Hepatitis C virus
proteolytic enzymes (alpha-1-antitrypsin and alpha-1-antichymotrypsin) may be helpful in the ✓ Human papilloma virus
diagnosis of malignant fibrohistiocytic sarcomas ✓ Cytomegalovirus
− An undifferentiated component of sarcoma may react only with vimentin ✓ Epstein-Barr virus
✓ Toxoplasma
3) VASCULAR TUMORS ✓ Pneumocystis carinii
✓ Helicobacter pylori
− Endothelial markers for vascular tumors (such as angiosarcomas) include Factor VII-related ✓ Cryptosporidium
antigen, CD31 and Ulex Europaeus 1 (UEA) ✓ Cryptococcus neoformans
4) MELANOMAS ✓ Histoplasma
✓ Entamoeba histolytica
− Melanocytes are derived from neural crest and will be reactive for S100 protein ✓ Mycobacteria
− the staining intensity for S100 is usually inversely proportional to the melanin content of the tumor For mycobacteria, immunohistochemical techniques are more sensitive, the results are obtained faster than
− Melanosome (HMB-45) is a widely used, highly sensitive and highly specific marker for the with tissue culture, and they are easier to read than acid-fast stains.
diagnosis of melanoma.
− Melan-A (MART-1) also encodes a melanoma-specific antigen that is present in normal pigmented CONTROLS
cells of skin and retina as well as in certain adrenocortical tumors
• It is essential to use positive and negative controls when processing tissue sections for
5) LYMPHOMAS immunohistochemistry, in order to:
✓ test for specificity of the antibodies involved
− LCA (leukocyte common antigen), also known as CD45 is the best screening marker for ✓ avoid misinterpretations due to false positive or false negative results
lymphoma
− for immunophenotypic subclassification of lymphoma, the most common markers used include • To be specific, the immunohistochemical technique must not cause staining in the absence of the
those for: primary antiserum.
✓ T cells (CD3, CD4, CDS) o The staining should be inhibited when the primary antibody is adsorbed by the relevant antigen
✓ B cells (CD19, CD20, CD23) prior to its use, but it should not be inhibited when the primary antibody is absorbed by other
✓ Reed-Sternberg cells (CD15, CD30) related or unrelated antigen.
✓ immunoglobulin light and heavy chains
POSITIVE CONTROL NEGATIVE CONTROL
CELL PROLIFERATION MARKERS It is always advisable to use, as positive control, a This can be done using a parallel section from the
section that is known and proven to contain the tissue, and either omitting the primary antibody
• Ki-67 (MIB-1) and PCNA (proliferating cell nuclear antigen) are the most common antigen in question because absence of staining in from the staining schedule or replacing the
immunohistochemical markers used to assess proliferation of tumor cells a test section does not necessarily mean that the specific primary antibody by an immunoglobulin
o Increased expression of these antigens is usually associated with greater aggressiveness and antigen is absent in the tissue being studied that is directed against an unrelated antigen.
higher likelihood of recurrence of metastasis.
 INTERNAL TISSUE CONTROL “BUILT-IN CONTROL”
• this eliminates the variable of tissue fixation between specimens and controls
• contains the target antigen, not only in the tissue elements under investigation but also in adjacent
normal tissue element
Ex: presence of S-100 protein in both melanoma and normal tissue elements, such as peripheral
nerves and melanocytes.
METHODS AND TECHNIQUES
CHROMOGENIC (BRIGHTFIELD) AND FLUORESCENCE DETECTION METHOD
• used in the determination of the presence and subcellular location of an increasing number of
proteins within a single biopsy
• Chromogenic Multiimmunohistochemical Staining is based on immunoenzymatic reaction with
chromogen and enzyme.
• Chromogenic IHC staining can generate dense deposits that are easy to detect but difficult to
quantitate
o this is due to nonlinear optical effects and low achievable dynamic ranges
• In facilitating chromogenic detection, the primary antibody, secondary antibody, or streptavidin is
conjugated to an enzyme.
► COMMONLY USED ENZYMES: Horseradish Peroxidase (HRP) and Alkaline Phosphatase,
convert:
3,3' diaminobenzidine (DAB) → brown end product
3-amino-9- ethylcarbazole (AEC) → red end product
When a soluble organic substrate is applied, the enzyme reacts with the substrate to generate an
insoluble colored product that is localized to the sites of antigen expression.
• Chromogenic detection is considered to be a more sensitive method than immunofluorescence.
o It requires only a typical light microscope unlike fluorescence microscopy which requires a
specialized light source and filter sets.
• Chromogenic detection, however, is less convenient because it includes more incubation and blocking
steps.
• Like immunofluorescence, it allows for the visualization of multiple antigens, but only if the antigens
are confined to different locations in the cell and tissue because overlapping colors may obscure results.
DAB Chromogenic Staining ADVANTAGE:
→ colored precipitate formed during the reaction between HRP and DAB is not sensitive to light and the
slides can be stored for many years.
ENZYME LABELLING
• Enzymes are widely used in immmunohistochemistry, and are usually incubated with a chromogen
using standard histochemical method to produce a stable colored reaction.
• Enzyme labeling of antibodies with horseradish peroxidase, followed by staining with appropriate
substrate or chromogen mixture (such as diaminobenzidine - DAB) will produce an insoluble dark
brown reaction end product, and allow labeled cells to be counterstained with hematoxylin and other
nuclear stains.
• The optimal incubation time for linking antibodies with peroxidase conjugates is 30-60 minutes at room
temperature.
DIRECT TECHNIQUE
• The traditional direct technique of doing immunohistochemistry is to conjugate the primary antibody
directly to the label (such as a fluorochrome or horseradish peroxidase)
• The method is no longer sufficiently sensitive for today's demands.
ADVANTAGE:
→ it is simple and quick, as it requires only one application of the reagent, followed by the appropriate
chromogen substrate solution
DISADVANTAGE:
→ it is less sensitive compared to indirect techniques that involve 2 or 3 stages of conjugation and staining.
» this carries the risk of not detecting small amounts of antigen that could be crucial in making the
diagnosis
ENHANCED POLYMER ONE-STEP STAINING (EPOS) METHOD
• Marketed by Dako A/S, is a new direct technique whereby a large number of primary antibody
molecules and peroxidase enzymes are attached to a dextran polymer "backbone" or "spine molecule"
ADVANTAGES:
→ reduced number of incubation steps of the staining protocol required, so that rapid staining is completed
in a single step within 10 minutes
→ in addition to an average of 70 molecules of enzyme, 10 molecules of antibody can be attached to the
spine molecule
→ it is more sensitive than the traditional direct technique, and is suitable for frozen section
immunohistochemistry
→ conjugation of both antirabbit and anti-mouse secondary antibodies renders the system useful for both
polyclonal and monoclonal antibodies.
→ these systems avoid the use of (strept) avidin and biotin, nonspecific staining that results from
endogenous biotin is eliminated.
DISADVANTAGE:
→ limited number of primary antibodies commercially available for this system.
 General EPOS Procedure (Peroxidase) PARAFFIN WAX SECTION IMMUNOPEROXIDASE TECHNIQUE

1. Quench for endogenous peroxidase activity (optional). • Paraffin Wax Section Immunoperoxidase Technique remains a STANDARD METHOD among most
2. Rinse with and place in wash buffer for 3 to 5 minutes. laboratories, especially in combination with frozen section processing for immunofluorescence of renal
3. Incubate with EPOS conjugate for 10 to 60 minutes. and skin biopsies.
4. Rinse with and place in wash buffer for 3 to 5 minutes. o Routinely fixed, paraffin-embedded specimens combine good morphology with localization of
5. Incubate with substrate-chromogen for 5 to 15 minutes. various cell and tissue markers
6. Counterstain (optional) and coverslip.
ADVANTAGES:
INDIRECT TECHNIQUE → it does not require an expensive fluorescence microscope
→ it can be adapted as a diagnostic procedure for formalin-fixed, paraffin embedded specimen
• it is a 2 or 3 step procedure that involves application of → immunolabeling can be correlated with morphology
the unconjugated primary antibody, followed by a
labeled antibody directed against the first antibody. DISADVANTAGE:
• it is relatively inexpensive, and is more sensitive → it is more time consuming than frozen-section immunofluorescence
than the traditional direct technique because several
secondary antibodies are likely to react with a number • In Renal and skin biopsies, proteolytic enzyme digestion is necessary, and is best achieved by using
of different epitopes of the primary antibody, thereby trypsin, chymotrypsin or protease
amplifying the signal as more enzyme molecules are
attached per target site. • Nonimmune serum is required to block nonspecific staining, especially when polyclonal antibodies
• Horseradish peroxidase is the most commonly used enzyme for indirect antibody enzyme-complex are employed.
techniques. o Avidin-biotin labeling, especially with peroxidase, is currently the most popular system used in
diagnostic laboratories
1) Two-Step Indirect Technique
• The most popular fixative for paraffin embedded sections is NEUTRAL BUFFERED FORMALIN.
a an unconjugated primary antibody first binds to the antigen o there may be shrinkage or distortion during fixation or subsequent paraffin-embedding, but
b enzyme-labeled secondary antibody directed against the primary antibody (now the antigen) is then generally, formalin-based fixatives are excellent for most immunostains
applied
c followed by the substratechromogen solution • Although some antigens are not well demonstrated after fixation in formaldehyde-based fixatives, many
can be demonstrated after the use of appropriate pretreatment methods, such as proteolytic enzyme
− If the primary antibody is made in rabbit or mouse, the secondary antibody must be directed digestion and/or antigen retrieval, particularly if polyclonal antisera are used
against rabbit or mouse immunoglobulins, respectively
− Cross-reactivity is eliminated by using preabsorbed secondary antiserum (i.e., antiserum • Mercuric chloride-based fixatives (formol sublimate and B5) have gained some popularity because
that has been absorbed with immunoglobulins from the species under investigation). they improve cytological preservation and minimize the distortion associated with formaldehyde-based
fixatives.
2) Three-Step Indirect Technique o They cause considerable hardening of tissue because of their coagulative properties, allowing thin
slices to be made.
− A second enzyme-conjugated antibody is added to the previously described indirect technique. o Thesy are particularly suitable for the demonstration of intracytoplasmic antigens
− The addition of a third layer of antibody serves to further amplify the signal, since more antibodies o B5 is widely recommended for the fixation of lymph node biopsies, both to:
are capable of binding to the previously bound secondary reagent. ✓ improve the cytological detail
✓ enhance immunoreactivity with the antiimmunoglobulin antisera used in phenotyping B
SOLUBLE ENZYME IMMUNE COMPLEX TECHNIQUES (UNLABELED ANTIBODY TECHNIQUES) cell lymphomas
► B5-fixed, paraffin embedded tissue sections show excellent results with cytoplasmic
• These techniques utilize preformed soluble enzyme-anti-enzyme immune complex. immunoglobulins
• The staining sequence involves the use of: » However, surface membrane immuno-globulin is not stained as readily.
✓ unconjugated primary antibody
✓ secondary antibody As a general rule, enzyme pretreatments do not improve, and may actually hinder immunostaining of
✓ soluble enzyme-anti-enzyme complex tissue fixed in mercuric chloride-based coagulative fixatives.
✓ substrate solution
• Alcohol is not widely used as a fixative for routine histological techniques because of its poor ADVANTAGE:
penetrating ability → lack of interference from endogenous peroxidase activity
o However, small pieces of tissue are fixed rapidly and show good cytological preservation
o Since alcohols are coagulant fluids and do not form additive compounds, they permit good Peroxidase-Antiperoxidase (PAP) Technique for Paraffin Sections
antibody penetration and do not block immunoreactive determinants
o Alcohol fixation is more commonly used in research laboratories where the size of specimens Solutions:
and handling requirements are different from those observed in routine histopathology Buffer Wash: 0.005M Tris buffered saline (TBS)
o It is also generally applied to frozen sections or smears. ✓ Tris-Buffered Saline Wash (0.005M TBS)
✓ Distilled water 1 liter
Preparing paraffin wax sections for immunostaining: ✓ Sodium chloride 8 gm
✓ TRIS (hydroxymethyl methylamine) 0.6 gm
1. Cut 3-S µm sections and place on cleaned glass slides. Vectabond or APES-coated slides may be ✓ M HCl 4.4 ml
used to assist with section adhesion.
2. Place sections at 60°C microwave oven overnight (if processing sections without adhesives) or at If necessary, adjust final pH to 7.6 with either 1 M HCl or 0.2 M Tris solution.
56°C for 1 to 2 hours (if Vectabond- or APEScoated slides are used). Heating on a hot plate at
high temperature is not recommended because it can be detrimental to the antigen. Substrate: DAB (Diaminobenzidine tetrahydrochloride)
3. Deparaffinize sections in xylene and bring to absolute alcohol. ✓ Tris-HCL Buffer (recommended for DAB)
4. Block endogenous peroxidase activity by incubating in 0.5% hydrogen peroxide in methanol for 10 ✓ 0.2 M Tris (containing 24.228 g/l) 12 ml
minutes. ✓ 0.1 M HCl 19 ml.
5. Rehydrate, wash well in running water and transfer to tris-buffered saline (TBS). ✓ Distilled water 19 ml.
6. To retrieve antigen:
a. Preheat slides at 37°C in distilled water. DAB Solution
b. Transfer to freshly prepared trypsin solution at 37°C for 15 to 120 minutes (depending on ✓ DAB 5 mg
specimen size, duration of fixation and rate of fixation), using 0.1% trypsin in 0.1% calcium ✓ Tris-HCI buffer (pH 7.6) 10 ml.
chloride in distilled water, adjusted to pH 7.8 using 0.1M NaOH and preheated at 37°C. ✓ H2O2 0.1 ml.
c. Terminate trypsin enzyme digestion by transferring the sections to cold running tap water.
(Freshly prepared and added just before use)

PEROXIDASE-ANTIPEROXIDASE (PAP) TECHNIQUE METHOD:

• An indirect antibody enzyme-complex technique 1. Bring sections to Tris-buffered saline (TBS).

where the soluble peroxidase-antiperoxidase complex 2. Drain off and wipe around section.
is bound to unconjugated primary antibody (e.g. rabbit 3. Incubate with 1:10 dilution of normal swine serum for 10 minutes. Drain, but do not wash off before
anti-human IgG) by a second layer of "bridging" applying primary antibody.
antibody usually a swine anti-rabbit antibody) that 4. Incubate in optimally diluted rabbit primary antibody for 30 to 60 minutes.
then binds to both the primary antibody and the rabbit 5. Gently wash in TBS.
PAP complex. 6. Incubate in optimally diluted swine anti-rabbit antibody for 30 minutes.
7. Gently wash in TBS.
• Horseradish Peroxidase is the most widely used 8. Incubate in optimally diluted rabbit peroxidase anti-peroxidase for 30 minutes.
enzyme for labeling 9. Gently wash in TBS.
o combining horseradish peroxidase with the most common chromogen – diaminobenzidine (DAB), 10. Incubate in freshly prepared DAB solution at room temperature until a dark brown reaction product
results in a stable, insoluble dark brown reaction end product when antigen is present in tissue is obtained, usually after 5 to 10 minutes. The reaction end-product resists alcohol dehydration and
o To block endogenous peroxidase activity, the sections are pre-incubated in absolute methanol clearing in xylene.
containing hydrogen peroxide 11. Rinse in TBS and wash in running water. 12. Counterstain in hematoxylin. 13. Dehydrate, clear and
mount.
• Alkaline phosphatase antibodies raised in mouse, by the same principle, can also be used to form
Alkaline Phosphatase-Anti-Alkaline Phosphatase Complexes (APAAP).
o APAAP technique is recommended for use on blood and bone marrow smears because of the
potential distraction of endogenous peroxidase activity on PAP staining.
o Endogenous alkaline phosphatase activity is usually blocked by adding levamisole to the
substrate solution.
Practical Considerations
1. Antigen-antibody reactions are reversible, and simple immune complexes formed on the tissue
may dissociate during the washing cycles used while performing immunohistochemistry.
2. Low salt concentrations as well as low temperatures will reduce the likelihood of weak staining
due to dissociation of already formed immune complex.
3. Monoclonal antibodies, compared to polyclonal antibodies, depend more on environmental factors
(pH and solute) for optimum performance. When using monoclonal antibodies, high salt
concentrations, high temperature and very low pH should be avoided during the washing of
specimens to avoid loss of staining due to weakening the antigen-antibody bond.
4. The recommendations for handling and storage given by the manufacturer on specification sheets
and on vial labels should always be followed in order to achieve optimal performance for the reagents
used in immunohistochemistry.
5. Refrigerators and freezers used for storage of immunochemicals should be monitored for accurate
and consistent temperatures.
6. Store most pre-diluted ("ready to use") antibodies, their conjugates, and monoclonal antibody
solutions at 2-8°C because freezing and thawing will reduce their effectiveness.
7. Concentrated protein solutions such as antisera and immunoglobulin fractions should be stored in
aliquots and frozen at -20°C or below to prevent cycles of repeated freezing and thawing.
8. Frozen protein solutions should be brought to room temperature slowly, and temperatures above
25°C should be avoided.
9. Reagent contamination can be avoided by the use of disposable clean pipette tips.
10. Higher concentrations of specific antibodies (and higher affinities) allow for the shortening of
incubation time.
11. The optimal incubation time for most primary antibodies is 20-30 minutes at room temperature (20-
22°C). Overnight (18 hour) incubation of sections at 4°C will increase the sensitivity of the procedure
with some monoclonal antibodies, particularly those against cell membrane antigens. To accomplish
this, adequate amounts of the primary antibody are applied, the section is covered with a cover glass,
and the slide is stored horizontally in a regular refrigerator.
12. It is possible to decrease the incubation time to 5-10 minutes by increasing the temperature to 37°C.
Slides incubated for extended periods or at 37°C should be placed in a humidity chamber to prevent
evaporation and drying of the tissue sections. Immunostaining in higher temperatures will have
unpredictable and sometimes cause false negative results.
13. Low levels of antigens may not be detected, and may result in false negative staining. Increasing the
concentration of the primary antibody or prolonging incubation with primary antibody overnight at 4°C
or at ambient temperature, can enhance staining.
14. Inhibiting endogenous enzyme activity, especially peroxidase and alkaline phosphatase, prior to
staining can eliminate false-positive reactions.
a. Endogenous enzyme peroxidase can be blocked by pre-incubating the sections in absolute
methanol containing 0.5% hydrogen peroxide for 10 minutes at room temperature.
b. Most endogenous alkaline phosphatase activity can be blocked by adding 1 mm concentration
of levamisole to the final incubating medium
15. Non-specific uptake of antigen can cause high levels of background staining. This can be due to
apparent affinity of collagen, reticulin and other tissue components for immuno-globulin, or due to
nonimmunological binding of specific immune sera within the tissue section.
16. Inadequate or delayed fixation may give rise to false-positive results due to passive uptake of plasma
proteins, including immunoglobulins by the cells.
17. Misinterpretation resulting from false-positive reactions can be avoided by using an anti-albumin
control.
18. In general, it is the first immune sera applied that gives rise to high levels of non-specific binding.
Background staining can be reduced by incubating the sections in an immunoglobulin that will not
react or interfere with the primary specific antiserum, such as normal whole serum from the species in
which the second (bridging) antibody is raised.
19. Nonspecific background staining can be reduced by adding a blocking serum to the diluted primary
antiserum, or by using primary antiserum at high dilutions, or by enzymatic digestion or by adding a
detergent such as Triton X.
20. When alkaline phosphatase serves as an enzyme label in the procedure, avoid using phosphate
buffers as they inhibit the activity of the enzyme.
21. Sodium azide, an antibacterial agent present in many commercially prepared buffers, can prevent
binding of the peroxidase enzyme to its substrate and inhibit color development. Its use in wash and
diluent buffers should be avoided.
22. Common chromogens for peroxidase are diaminobenzidine (DAB) and aminoethylcarbazole
(AEC), both of which should be made fresh immediately before use. Failure to add hydrogen peroxide
to either of these solutions is a common oversight, particularly or beginners, and results in total lack of
staining of all slides, including positive controls.
23. Etching a circle around the tissue section with a diamond pen will prevent diffusion of reagent over
the entire slide, thereby saving precious antibody.
Blocking of Unwanted Non-specific Staining
• Unwanted non-specific staining, a common problem in immunohistochemistry experiments, generally
occurs when there is binding of the primary antibody to amino acids other than those within the desired
epitope of the antigen.
• Nonspecific binding causes high background staining that can mask the detection of the target antigen.
• Sources of unwanted non-specific staining include:
✓ endogenous enzymes or fluorochromes
✓ endogenous biotin
✓ endogenous antibody binding activity
✓ cross reactivity of the secondary reagents with endogenous proteins
o They result in high background causing difficulties in visualizing the antigen of interest in its
appropriate cellular location
• The use of a blocking reagent prior to incubation of the sample with the primary antibody can effectively
block nonspecific staining interactions.
• Common blocking buffers include:
✓ normal serum
✓ non-fat dry milk
✓ BSA or gelatin
✓ commercial blocking buffers with proprietary formulations
AVIDIN-BIOTIN COMPLEX (ABC) TECHNIQUES
• It uses AVIDIN (derived from egg white) because of its marked affinity for biotin, a low molecular weight
vitamin that can be easily conjugated to antibodies and enzyme markers
o Variants of the avidin-biotin system include:
✓ peroxidase and alkaline phosphatase, either directly bound to avidin or streptavidin (a
similar molecule extracted from the culture broth of the bacterium Streptomyces avidini)
 ✓ Enzymes are biotinylated and the avidin-binding sites are occupied by the biotinylated label, • The staining sequence consists of:
forming the avidin-biotin complex 1. primary rabbit (or mouse) antibody
2. biotinylated anti-rabbit (or anti-mouse) immunoglobulin
• The basic sequence of staining consists of: 3. streptavidin-enzyme conjugate.
1. primary antibody
2. biotinylated secondary antibody The color reaction is then developed with the appropriate substrate/ chromogen, such as
3. followed either by the preformed (strept) avidin- horseradish peroxidase.
biotinenzyme complex of the avidin-biotin complex
(ABC) technique or by the enzyme labeled METHOD
streptavidin
1. Paraffin section or frozen section to water and rinse in PBS-Tween 20 times for 2 minutes each
• Formation of ABC complex requires that the solutions of the time.
(strept)avidin and biotinylated enzyme are mixed and 2. Perform antigen retrieval if necessary.
prepared at least 30 minutes before use. All incubations are carried out at room temperature. 3. Incubate sections in normal serum – species same as secondary antibody. Note: since this
protocol uses avidin-biotin detection system, avidin/biotin block may be needed based on tissue
METHOD type.
4. Incubate sections with primary antibody at appropriate dilution for 1 hour at room temperature or
1. Paraffin section or frozen section to water and rinse in PBS-Tween 20 twice for 2 minutes each overnight. No serum blocking is needed if antibody diluent is used.
time. 5. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time.
2. Perform antigen retrieval if necessary. 6. Incubate sections in peroxidase blocking solution for 10 minutes at room temperature. Note: For
3. Incubate sections in normal serum – species the same as secondary antibody. Note: Since this acetone fixed frozen sections, perform this peroxidase blocking step using 0.3% H2O2 in methanol
protocol uses avidin-biotin detection system, avidin/biotin block may be needed based on tissue prior to primary antibody incubation to avoid tissue destruction.
type. 7. Rinse with PBS-Tween 20 buffer 3 times for 2 minutes each time.
4. Incubate sections in primary antibody at appropriate dilution for 1 hour at room temperature or 8. Incubate sections in Biotinylated secondary antibody in PBS for 30 minutes at room temperature.
overnight. Note: No serum blocking is needed if antibody diluent is used. 9. Rinse with PBS-Tween 20 buffer 3 times for 2 minutes each time.
5. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 10. Incubate sections in HRP-Streptavidin solution for 30 minutes at room temperature.
6. Incubate sections in peroxidase blocking solution for 10 minutes at room temperature. 11. Rinse with PBS-Tween 20 buffer for 3 times for 2 minutes each time.
Note: For acetone fixed frozen sections, perform this peroxidase blocking step using 0.3% H2O2 in 12. Incubate sections in peroxidase substrate solution.
methanol prior to primary antibody incubation to avoid tissue destruction. 13. Rinse with PBS-Tween 20 buffer for 3 times for 2 minutes each time.
7. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 14. Counterstain with hematoxylin.
8. Incubate sections in biotinylated secondary antibody in PBS for 30 minutes at room temperature. 15. Rinse in running tap water for 2-5 minutes.
9. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 16. Dehydrate through 95% ethanol for 1 minute, and then 100% ethanol 2 times for 3 minutes each
10. Incubate sections in ABC-Peroxidase Solution for 30 minutes at room temperature. time.
11. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 17. Clear in xylene 2 times for 5 minutes each time.
12. Incubate sections in peroxidase substrate solution. 18. Coverslip.
13. Rinse in PBS-Tween 30 buffer 3 times for 2 minutes each time.
14. Counterstain with counterstain solution. IMMUNOFLUORESCENCE (IF) METHOD
15. Rinse in running tap water for 2-5 minutes.
16. Dehydrate through 95% ethanol for 1 minute, and 100% ethanol 2 times for 3 minutes each time. • An emerging prime alternative to chromogenic approaches to IHC as it has the ability to:
17. Clear in xylene 2 times for 5 minutes each time. ✓ generate highresolution images for protein localization studies
18. Coverslip. ✓ quantitate the fluorescent signal

LABELED STREPTAVIDIN BIOTIN TECHNIQUE (LSAB PROCEDURE) • Immunofluorescent methods are extensively used to detect antibodies, particularly for the diagnosis of:
✓ glomerular disease in frozen sections of renal biopsies
• A Labeled Avidin-Biotin (LAB) Method has been recently introduced and is found to be 4-8 times more ✓ skin biopsies of patients with systemic lupus and vasculitis, to examine the pattern of deposition of
sensitive than the old ABC method immunoglobulins

• Avidin has now been largely replaced by the use of streptavidin, leading to the labeled streptavidin-biotin • In addition, technical advances in microscope development and fluorophore have widened the selection
(LSAB) method. of colors to use for both single- and multi-color fluorescence microscopy greater than ever.
• This method is used in the evaluation of: 5. Incubate in freshly prepared DAB solution at room temperature until a dark brown reaction
✓ cells in suspension product is obtained, usually after 5-10 minutes. The reaction end-product resists alcohol
✓ cultured cells, tissue, beads and microarrays dehydration and clearing in xylene.
6. Rinse in TBS and wash in running water.
• Its practical application in laboratory include: 7. Dehydrate through 95% ethanol for 1 minute, and then 100% ethanol 2 times for 3 minutes
1. the analysis of antigens in fresh, frozen or fixed tissues, sub-cellular localization of antigens in each time.
tissue culture monolayers and observation of bacterial or parasitic specimens 8. Clear in xylene 2 times for 5 minutes each time.
2. detection and localization of the presence or absence of specific DNA sequences on 9. Coverslip.
chromosomes
3. defining the spatial-temporal patterns of gene expression within cells/tissues. RESULTS:

• Fluorescence and Phosphorescence are both types of luminescence Apple-green fluorescence when fluorescein is used as fluoro-chrome; Orange-red fluorescence with
o When molecules with luminescent properties absorb light, they emit light of a different wavelength. rhodamine conjugates

• In the immunofluorescence method, antibodies are chemically conjugated to fluorescent dyes such as:
✓ fluorescein isothiocyanate
✓ tetramethyl rhodamine isothiocyanate

These labeled antibodies bind (directly or indirectly) to the antigen of interest which allows for
antigen detection through fluorescence techniques.

• The fluorescence can then be quantified using:

✓ flow cytometer
✓ array scanner or automated imaging instrument NOTES:
✓ visualized using fluorescence or confocal microscopy
✓ The slides may be stored at 4°C for one year, but may show decreased fluorescent staining.
• Successful immunofluorescent techniques depend on: ✓ Avoid direct sunlight on the slides at all times.
✓ adequate preservation of substrate antigens ✓ Cover slip should not be moved or maneuver when mounting, to prevent distortion of the
✓ adequacy of antibody conjugate tissue.
✓ careful staining and incubation procedures ✓ The tissue sections must be kept moist at all times to prevent artefactual staining.
✓ quality of the fluorescence microscope. ✓ Conjugates prepared from poor quality antibody tend to produce inferior results, often with
weak positive results against high levels of background staining.
1) Direct Immunofluorescence Technique for Solid Tissue Biopsies
2) Indirect Immunofluorescence Technique
− this is usually performed on thin (2 to 5 µm) cryostat sections of fresh unfixed material, mounted on
slides that have been previously coated with gelatin adhesive or poly-L-lysine (supplied by Sigma) − mainly used for the detection of autoantibodies in the patient's serum, including the:
at 1:10 dilution. ✓ anti-nuclear antibody (ANA)
− the tissue is reacted directly with a fluorescein-conjugated antibody specific for the material being ✓ anti-mitochondrial antibody (AMA)
sought within the tissue ✓ liver-kidney microsomal antibody

METHOD 3) Frozen Section Immunofluorescence

1. Bring sections to Tris-buffered saline (TBS), drain off, and incubate in non-immune serum. − a relatively simple, rapid and sensitive technique that is easily reproducible, particularly among
2. Drain off and wipe around section. histopathologists who are experienced with fluorescent antibody techniques
3. Incubate in optimally diluted peroxidase-labeled primary antibody for 1-15 hours (traditional − however, often fades within days after the sections have been immunostained
direct method) at ambient temperature or 4°C; or in EPOS peroxidase pre-diluted antibody − it requires the use of a costly fluorescence microscope, which can be a major drawback.
(DAKO) for 1-2 hours at ambient temperature (enhanced polymer one-step method). − Cryostat sections give much better antigen preservation than paraffin sections
4. Gently wash in TBS.
 − Fresh tissue should be frozen immediately and rapidly
» A 1.0 x 1.0 x 0.3 cm block of fresh tissue can be snap-frozen by immersing it directly in liquid
nitrogen, although a mixture of isopentane and liquid nitrogen will result in a more
uniform freezing of tissue and better preservation of histomorphology.
» OCT can be used as a supporting media to facilitate preparation of good quality frozen
sections
▪ prolonged storage of OCT-embedded frozen tissue will cause:
✓ gradual loss of cellular antigens
✓ snap-frozen fresh tissue
• Pre-treatment digestion with protease enzyme is required for all cross linked samples to remove
proteins and make the target more accessible to the probe
o because of unavoidable variations in tissue fixation and processing, the use of an endogenous
positive control probe is absolutely essential
• Heat and formamide break hydrogen bonds, are the commonly used denaturants.
o when complementary strands from two different sources are mixed and the denaturants are
removed, some of the double-stranded structures will be composed of one strand from each
source, forming molecules known as "hybrids"

Without supporting media, can be stored at -70°C for long periods of time without In a hybridization assay, the two sources are the target (sample) and the probe nucleic acids
appreciable antigen loss
• A probe is simply a known fragment of nucleic acid with a label that can be detected in some fashion
− Acetone is generally used as fixative to preserve the antigen, to: o it forms a hybrid molecule with a sample that contains nucleic acids complementary to its
✓ destroy harmful infective agents sequence
✓ allow a wide range of primary antibodies to be employed without destroying many of the
epitopes The process of searching a sample for specific nucleic acid sequences is "hybridization reaction"

» Sections are fixed in absolute acetone at room temperature for 30 minutes, and air-dried for a
few minutes prior to immunostaining.
» Cell surface antigens are best preserved in sections fixed briefly in cold acetone (5 minutes at
4°C)
− Good results are also obtained in frozen sections fixed for a few minutes in ethanol, formalin and
picric acid paraformaldehyde
− Between each step of the staining technique, sections require several brief washing with Tris
Buffered Saline (TBS) to prevent one reagent from contaminating another
− Thin sections and extended drying period prevent the artifacts often seen in frozen-section
immunostains of lymphoid tissues fixed in acetone
» Extending the drying period to 48 hours will usually result in improved morphology.
SLIDE PREPARATION OF FROZEN SECTIONS
• Majority of the probes now available are produced either by: Recombinant Nucleic Acid Technology
or Chemical Synthesis
o Cloned probes consist of a known segment of DNA inserted into a plasmid vector that is
propagated by growth in bacterium, resulting in a double-stranded DNA probe which must be
denatured before use
o Other plasmid vectors contain RNA promoter regions that permit generation of single-strand RNA
probes, RNA probes require careful handling and storage to prevent degradation.
» RNA is much more unstable than DNA, and enzymes that digest RNA (known as RNase)
are virtually present everywhere.
The use of RNA probes therefore dictate use of sterile technique and preparation of
reagents and glassware to remove RNase
• All hybridization assays require the probe and the sample nucleic
acid to be mixed under conditions that will allow complementary
base-pairing as well as a method to detect that hybridization has
occurred.

1. Snap-freeze fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, • Detection of radio-labeled probes is usually achieved with
embedded in OCT compound in cryomolds. AUTORADIOGRAPHY
2. Store frozen blocks at -80oC.
3. Cut 4-8 m thick cryostat sections. • Non-isotopic labeling has been initially achieved through the
4. Mount cryostat sections on either super-frost plus slides or gelatin coated slides. production of biotin-labeled analogue of deoxyuridine
5. Store slides at – 80oC. triphosphate
6. Prior to staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone o Biotin itself cannot generate signals, so that polynucleotides
for 5 minutes. Air dry for 30 minutes. with biotin incorporated into their structure are detected
7. Wash in PBS. indirectly through high-affinity interaction with avidin or streptavidin chemically linked or complexed
to a colorimetric enzyme or fluorescence tags.
IN-SITU HYBRIDIZATION
• Alkaline phosphatase systems with tartrazine substrates are reported to yield good results.
• It is based on the specificity of the interaction of a probe with the target nucleic acid, rather than the
target protein or immunogen. • The lack of commercial reagents is one of the most significant factors limiting the diagnostic application
of in-situ hybridization in many laboratories.
• Formalin fixed paraffin-embedded material appears to be the best choice in a diagnostic setting.