Immunohistochemistry PDF

CHAPTER 22 IMMUNOHISTOCHEMISTRY Jocelyn H. Bruce and Marc-Eli Faldas Immunohistochemical techniques are now routinely used for the identification of specific or highly selective cellular epitopes or antigens in frozen or paraffin-embedded tissues. Immunocytochemistry can al...

CHAPTER 22 IMMUNOHISTOCHEMISTRY Jocelyn H. Bruce and Marc-Eli Faldas Immunohistochemical techniques are now routinely used for the identification of specific or highly selective cellular epitopes or antigens in frozen or paraffin-embedded tissues. Immunocytochemistry can also be used to detect organisms in cytologic preparations such as fluids, sputum samples, and fine needle aspirates. These immunologic techniques make use of antigen​- antibody interactions, whereby the site of antigen binding is demonstrated by direct labeling of the antibody, or by means of a secondary labeling method. Antibodies belong to the class of serum proteins known as immunoglobulins. IgG is the most commonly used antibody for immunocytochemistry. An epitope is the structural part of the antigen that reacts with an antibody. Immunohistochemistry (IHC) combines anatomical, immunological and biochemical techniques to identify discrete tissue components by the interaction of target antigens with specific antibodies tagged with a visible label. IHC makes it possible to visualize the distribution and localization of specific cellular components within cells and in the proper tissue context. While there are multiple approaches and permutations in IHC methodology, all of the steps involved are separated into two groups: sample preparation and labeling. IHC is used for disease diagnosis, drug development and biological research. Using specific tumor markers, physicians use IHC to diagnose a cancer as benign or malignant, determine the stage and grade of a tumor, and identify the cell type and origin of a metastasis to find the site of the primary tumor. IHC is also used in drug development to test drug efficacy by detecting either the activity or the up- or down-regulation of disease targets. Immunofluorescence is often, but not always performed on frozen tissue due to the high background auto-fluorescence seen in formalin fixed paraffin embedded tissue. Samples are prepared on individual slides, or multiple samples can be arranged on a single slide for comparative analysis, such as with tissue microarrays. IHC slides can be processed and stained manually, while technological advances now provide automation for high-throughput sample preparation and staining. Samples can be viewed by either light or fluorescence microscopy, and advances in the last 15 years have improved the ability to capture images, quantitate mult-0parametric IHC data and increase the collection of that data through high content screening. Polyclonal Antibodies Polyclonal antibodies are produced by immunizing an animal with a purified specific molecule (immunogen) that contains the antigen of interest, and collecting immunoglobulin-rich serum after the animal has produced humoral antibody against the antigen. The most frequently used animal for the production of polyclonal antibodies is the rabbit, followed by goat, pig, sheep, horse, guinea pig and others. Because polyclonal antibodies are produced by different cells of the animal, they are immunochemically not identical to each other, and they react with various epitopes on the antigen against which they are raised. Some of the polyclonal antibodies may cross-react with other molecules and cause non- specific staining, requiring their purification by absorption with the appropriate antigen, or antibody dilution to eliminate the unwanted reaction. Monoclonal Antibodies Animals immunized with the specific immunogen will produce numerous clones of plasma cells (polyclonal) that in turn will produce the antibody. Monoclonal antibodies are the products of an individual clone of plasma cells. Hybridoma and cloning techniques have been developed to produce monoclonal antibodies that do not cross-react with other molecules. Antibodies from a given clone are immunochemically identical and react with a specific epitope on the antigen against which they are raised. Mice are currently used almost exclusively for the production of monoclonal antibodies. Propagation can be carried out in culture medium or by transplantation of the hybridoma into the peritoneal cavity of syngeneic mice from where the antibodies are harvested. This has dramatically increased the quantities and number of specific monoclonal antibodies available for immunohistochemistry. Preparing tissue for immunohistochemistry In certain instances, the tissue must be prepared as a cryostat section and fixed for a few seconds in absolute methanol or acetone, to preserve immunological activity and prevent destruction of some of the labile antigenic sites. However, immunofluorescence and immuno-peroxidase techniques may also be done on formaldehyde-fixed and paraffin embedded sections. Many masked antigens can now be retrieved in routinely processed tissue by (1) proteolytic enzyme digestion, (2) microwave antigen retrieval, (3) microwave and trypsin antigen retrieval or (4) pressure cooker antigen retrieval. Proteolytic Enzyme Digestion Formalin fixed paraffin sections are usually pre-treated with proteolytic enzymes to break down formalin cross-linking, unmask and allow certain antigenic sites to be exposed. Proteolytic enzyme digestion is especially useful for demonstrating heavy chain immunoglobulins, complement and specific antigens (such as cytokeratin) in formalin-fixed paraffin-embedded biopsies. The most common enzymes used are trypsin and protease. Before pre​- treatments are employed, the sections are deparaffinized, taken to alcohol and, in the case of peroxidase labeling, treated with 0.5% methanol in hydrogen peroxide for 10 to 15 minutes to destroy endogenous peroxidase activity. The slides are then washed in running water and taken to distilled water. The trypsin method uses 0.1% trypsin in 0.1 % calcium chloride in distilled water, adjusted to pH 7.8 with sodium hydroxide, and preheated at 37°C. The slides are also preheated at 37°C in distilled water before placing in freshly prepared trypsin solution. After a predetermined period of time, the slide is transferred to cold running water to terminate enzyme digestion. The protease method uses 0.05 to 0.1% protease in distilled water, adjusted to pH 7.8 with sodium hydroxide. 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. Paraffin Sections 1. Deparaffinize sections in xylene 2 times for 5 minutes each time. 2. Hydrate with 100% ethanol 2 times for 3 minutes each time. 3. Hydrate with 95% ethanol for 1 minutes. 4. Rinse in distilled water. 5. Follow procedure for pretreatment as required. Pre-treatment of Tissue Sections Antigenic determinants masked by formalin-fixation and paraffin- embedding often may be exposed by epitope unmasking, enzymatic digestion or saponin, etc. Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded. Procedure 1. Rinse sections in PBS-Tween 2 times for 2 minutes each time.  2. Serum Blocking: Incubate sections with normal serum block – species same as secondary antibody, for 30 minutes to block non-specific binding of immunoglobulin. 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 antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C. 4. Rinse in PBS-Tween 20. 5. Peroxidase Blocking: Incubate sections in peroxidase blocking solution for 10 minutes at room temperature. 6. Rinse in PBS-Tween 20. 7. Secondary Antibody: Incubate sections with biotinylated secondary antibody at appropriate dilution in PBS for 30 minutes at room temperature. 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. 10. Rinse in TBS 3 times for 2 minutes each time. 11. Chromogen/Substrate: Incubate sections in DAB solution for 1-3 minutes. 12. Rinse in PBS-Tween 20 2 times for 2 minutes each time. 13. Counterstain if desire. 14. Rinse in distilled water. 15. Dehydrate through 95% ethanol for 2 minutes, then 100% ethanol for 2 times 3 minutes each time. 16. Clear in xylene. 17. Coverslip with mounting medium. Heat-induced epitope retrieval (HIER) Heat-induced epitope retrieval (HIER), a simple laboratory technique that has become an essential part of many immunohistochemistry and in situ hybridization procedures, is a pretreatment method used to improve staining results. Heat, coupled with specific buffered solutions, is utilized to recover antigen reactivity in formalin fixed paraffin embedded tissue. It reverses the formaldehyde mediated chemical modifications of the antigen through either of the following processes. First and foremost, thermal energy breaks the crosslinks that bind surrounding proteins or peptides to the antigen which lead to the “opening” or “unmasking” the epitope. And, second, thermal energy removes bound calcium ions from the sites of cross-links since several HIER buffers, such as EDTA and citrate, act as calcium chelators. HIER heating sources include the microwave, vegetable steamer, pressure cooker and water bath. In general, the higher the temperature of the HIER solutions, the more effective the recovery of the epitope is. While each of these heating sources is suitable for HIER, there are advantages as well as drawbacks associated with each source. For example, often, the microwave distribution of heat within a microwave is uneven or inconsistent which results in a lack of reproducibility as to staining intensities. In contrast, the pressure cooker, steamer and water bath produce uniform and consistent heat distribution. However, while the higher temperatures produced by the pressure cooker are advantageous since, in a short period of time, an effective recovery of epitope reactivity can be readily achieved, damage or distortion to the morphology of connective tissues can also occur. Heat-Induced Epitope Retrieval (HIER) www.rndsystems.com Reagents Required for Heat-induced Epitope Retrieval: 10 x Antigen Retrieval Solution: o Antigen Retrieval Reagent-Basic o Antigen Retrieval Reagent-Acidic o 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 92-95°C Procedure: 1. Make working dilutions by mixing 1 part of 10X 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. Microwave Antigen Retrieval Microwave antigen retrieval is a relatively new technique that involves the boiling of formalin-fixed deparaffinized sections in certain solutions, such as 0.01 M-citrate buffer (pH 6.0), EDTA at pH 8.0 or 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. Antibodies 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, are demonstrated well on paraffin sections after heat pre-treatment. Most antigen retrieval methods apply temperatures near the boiling point of water. The optimal length of exposure to heat may vary from 10 to 60 minutes and depends to some extent on the length of formalin fixation. The most satisfactory time period appears to be 20 minutes 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), or dipping Vectabond-coated slides in I0% formol saline for 1 to 2 minutes and 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 hr. of fixation time. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr. in the formalin-fixed tissue. Bouin's and B-5 tissues give consistent results only after 6 hr. 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. Pressure Cooking Antigen Retrieval Pressure cooking antigen retrieval is another alternative that appears to be less time consuming and allows for more consistent recovery of many antigens, compared to large batch microwave oven technique. 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. Antigens Primary antibodies against numerous antigens are now available in the market, and are widely used for diagnosis of tumors, determination of tumor type, the evaluation of prolife-ration potential, identification of infectious agents, prognostic and therapeutic implications, and many other aspects of diagnostic pathology. Epithelial tumor markers: 1. Keratin is a highly sensitive marker for epithelial cells, and is present in epithelial tumors (carcinoma). Certain non-epithelial tumors (such as mesotheliomas and non-seminomatous germ cell tumors) also stain positive for keratin, and may be distinguished from carcinoma by applying an additional panel of antibodies. a) CK7 (Cytokeratin 7) is more frequently found in carcinomas of the lung, breast, uterus and ovaries (serous tumors). These tumors are typically negative for CK20. b) CK20 (Cytokeratin 20) is more common in carcinomas of the colon and stomach. These tumors are usually negative for CK7. c) Transitional cell carcinomas of the bladder and mucinous ovarian tumors are usually positive for both CK7 and CK20. d) Renal cell carcinomas, hepatocellular carcinomas, prostatic adenocarcinomas, thyroid carcinomas and squamous cell carcinomas (skin, lung and esophagus) are usually negative for either CK7 or CK20. 2. EMA (Epithelial membrane antigen) is a high molecular weight protein that is helpful in determining the site of tumor. It is positive for adenocarcinomas of the breast, lung and kidneys but more often non​reactive for hepatocellular carcinomas, adrenal carcinomas or embryonal carcinomas, and negative for non-epithelial tumors (sarcomas, lymphomas, melanomas) and other tumors (meningiomas, mesotheliomas, anaplastic large cell lymphomas, and plasma cell tumors). 3. CEA (Carcinoembryonic antigen) is an oncofetal antigen that is present in carcinomas of the gastrointestinal tract, pancreas, lung, breast, ovary, uterus and cervix. It is especially useful for differentiating between adenocarcinoma (CEA-positive) and mesothelioma (CEA-negative). Prostate, thyroid and renal carcinomas are usually non-reactive to CEA. 4. TTF-1 (Thyroid transcription factor-1) is useful in distinguishing lung adenocarcinomas from mesotheliomas. It is positive in thyroid, lung and neuroendocrine tumors (medullary thyroid carcinomas, carcinoid tumors and small cell tumors of the lung). 5. PSA (Prostate specific antigen) is extremely useful in the diagnosis of prostatic adenocarcinoma. It is also positive in certain pancreatic and salivary gland tumors. Intermediate Filament Markers 1. Actin is a contractile intermediate filament protein present in muscle and some non-muscle tissue. It is a sensitive marker for muscle differentiation and can be used to identify tumors derived from smooth, skeletal and cardiac muscle. 2. Vimentin is a 57kD intermediate filament that is present in normal mesenchymal cells and their neoplastic 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 is a 53 kD intermediate filament expressed by smooth and striated (skeletal and cardiac) muscle. It is considered to be highly specific for myogenic tumors, including leiomyoma (smooth muscle tumor) and rhabdomyosarcoma (skeletal muscle tumor). It is also used to demonstrate the myogenic component of mixed tumors (i.e., carcinosarcomas or malignant mixed mesodermal tumors). 4. Glial fibrillary acidic protein (GFAP) is a 51 kD intermediate filament protein expressed by central nervous system glial cells, particularly astrocytes. It is most widely used to confirm the diagnosis of astrocytoma (but may also be present in certain cases of ependymomas, oligodendrogliomas and medulloblastomas). Non-CNS tumors (meningiomas, metastatic carcinomas and lymphomas) stain negative for GFAP. 5. Neurofilament (NF) is expressed in cells of neural origin, particularly neurons, neuronal processes, peripheral nerves, sympathetic ganglia, adrenal medulla and neuroendocrine cells. Tumors that show neuronal or neuroendocrine differentiation (e.g., neuroblastomas, ganglioneuromas, neuromas, chemodectomas, and pheochromocytomas) will stain positive for neurofilament. 6. S-100 protein is a low molecular weight calcium-binding protein that is expressed in CNS glial cells, Schwann cells, melanocytes, histiocytes, chondrocytes, skeletal and cardiac muscle, myoepithelial cells and some epithelial cells of breast, salivary and sweat gland epithelium. Neuroendocrine markers: 1. Neuron-specific enolase (NSE) is an isoenzyme marker whose presence in tissue provides strong evidence of neural or neuroendocrine differentiation. 2. Chromogranin is found in the neural secretory granules of endocrine tissues, and is recognized as a marker for neuroendocrine differentiation. Immunoreactivity is typically granular and its distribution is similar to that seen with silver staining methods such as Grimelius stain. A combination of keratin and chromogranin positivity is typical of neuroendocrine carcinoma. Chromogranin positivity with a negative keratin stain is typical of paraganglioma. 3. Synaptophysin is a 38 kD transmembrane protein associated with presynaptic vesicles of neurons. It has been identified in normal neurons and neuroendocrine cells. Germ cell tumor markers 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 classification, the following germ cell tumor markers are used: 1. HCG (Human chorionic gonadotropin) is synthesized by placental syncytiotrophoblasts, and is a marker for choriocarcinoma. 2. APP (Alpha-fetoprotein) is synthesized by normal liver  hepatocytes, and is used as a marker for endodermal sinus tumors showing yolk sac differentiation. Embryonal carcinomas and teratomas containing these elements, as well as hepatocellular carcinomas will also stain positive for APP. 3. PLAP (Placenta-like alkaline phosphatase) is produced by the placental syncytiotrophoblasts in late pregnancy, and is used as a marker for germ cell tumors, particularly germinomas. Most embryonal carcinomas, choriocarcinomas and endo-dermal sinus tumors will also stain positive for this antibody. PLAP is positive in majority of seminomas. Mesenchymal tumor markers 1. Myogenic tumors - Tumors of skeletal muscle origin are positive for muscle-specific actin and desmin and/or other muscle markers such as myo-D1, myoglobin and myogenin. 2. Fibrohistiocytic tumors - The use of histiocytic markers such as CD68, or FAM 56, combined with more nonspecific proteolytic enzymes such as alpha-1-antitrypsin and alpha-1-antichymotrypsin may be helpful in the diagnosis of malignant fibrohistiocytic sarcomas. An undifferentiated component of sarcoma may react only with vimentin. 3. Vascular tumors - Endothelial markers for vascular tumors (such as angiosarcomas) include Factor VII-related antigen, CD31 and Ulex Europaeus 1 (UEA). 4. Melanomas - Melanocytes are derived from neural crest and will be reactive for S100 protein. The intensity of staining for S100 is usually inversely proportional to the melanin content of the tumor. Melanosome (HMB-45) is a widely used, highly sensitive and highly specific marker for the diagnosis of melanoma. Melan-A (MART-1) also encodes a melanoma-specific antigen that is present in normal pigmented cells of skin and retina as well as in certain adrenocortical tumors. 5. Lymphomas - The best screening marker for lymphoma is LCA (leukocyte common antigen), also known as CD45. For immunophenotypic subclassification of lymphoma, the most common markers used include those for T cells (CD3, CD4, CDS), B cells (CD19, CD20, CD23), Reed-Sternberg cells (CD15, CD30), and immunoglobulin light and heavy chains. Cell Proliferation Markers Ki-67 (MIB-1) and PCNA (proliferating cell nuclear antigen) are the most common immunohistochemical markers used to assess proliferation of tumor cells. Increased expression of these antigens is usually associated with greater aggressiveness and higher likelihood of recurrence of metastasis. Cancer-associated genes 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 cellular oncogenes, including c-erbB-2, c-myc and ras have been found to be activated in cancer, particularly of the breast. Infectious Agent Markers Antigenic markers are now available for a number of infectious agents, including hepatitis A virus, hepatitis B surface and core antigens, hepatitis C virus, human papilloma virus, cytomegalovirus, Epstein-Barr virus, toxoplasma, pneumocystis carinii, helicobacter pylori, cryptosporidium, cryptococcus neoformans, histoplasma, entamoeba histolytica, and mycobacteria. For mycobacteria, immunohistochemical techniques are more sensitive, the results are obtained faster than with tissue culture, and they are easier to read than acid-fast stains. Controls It is essential to use positive and negative controls when processing tissue sections for immunohistochemistry, in order to test for specificity of the antibodies involved, and to avoid misinterpretations due to false positive or false negative results. To be specific, the immunohistochemical technique must not cause staining in the absence of the primary antiserum. The staining should be inhibited when the primary antibody is adsorbed by the relevant antigen prior to its use, but it should not be inhibited when the primary antibody is absorbed by other related or unrelated antigen. 1. Positive Control: It is always advisable to use, as positive control, a section that is known and proven to contain the antigen in question because absence of staining in a test section does not necessarily mean that the antigen is absent in the tissue being studied. 2. Negative Control: This can be done using a parallel section from  the tissue, and either omitting the primary antibody from the staining schedule or replacing the specific primary antibody by an immunoglobulin that is directed against an unrelated antigen. 3. Internal Tissue Control: Also named as "built in" control, this eliminates the variable of tissue fixation between specimens and controls but it contains the target antigen, not only in the tissue elements under investigation, e.g. tumors, but also in adjacent normal tissue element. One example is the presence of S-100 protein in both melanoma and normal tissue elements, such as peripheral nerves and melanocytes. Chromogenic Method Chromogenic (brightfield) and fluorescence detection techniques are used in the determination of the presence and subcellular location of an increasing number of proteins within a single biopsy. Chromogenic multi- immunohistochemical 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, because of nonlinear optical effects and low achievable dynamic ranges. In facilitating chromogenic detection, the primary antibody, secondary antibody, or streptavidin is conjugated to an enzyme. Horseradish peroxidase (HRP) and alkaline phosphatase, which convert 3,3' diaminobenzidine (DAB) and 3-amino-9- ethylcarbazole (AEC), into brown and red end products, respectively are commonly used enzymes. 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. 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. An advantage of DAB chromogenic staining is that the 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 Labeling  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 to 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 main advantage of using directly labeled antibody is that it is simple and quick, because it requires only one application of the reagent, followed by the appropriate chromogen substrate solution. However, 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. The method is no longer sufficiently sensitive for today's demands. Enhanced Polymer One-Step Staining (EPOS) method The 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". The chief advantage of the EPOS technology is the 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 anti- rabbit and anti-mouse secondary antibodies renders the system useful for both polyclonal and monoclonal antibodies. Because these systems avoid the use of (strept) avidin and biotin, nonspecific staining that results from endogenous biotin is eliminated. The main disadvantage is the limited number of primary antibodies commercially available for this system.  Fig. 22-1. Immunohistochemistry - Direct Technique General EPOS Procedure (Peroxidase) 1. Quench for endogenous peroxidase activity (optional). 2. Rinse with and place in wash buffer for 3 to 5 minutes. 3. Incubate with EPOS conjugate for 10 to 60 minutes. 4. Rinse with and place in wash buffer for 3 to 5 minutes. 5. Incubate with substrate-chromogen for 5 to 15 minutes. 6. Counterstain (optional) and coverslip. Indirect Technique The indirect technique of immunohistochemistry is a 2 or 3 step procedure that involves application of the unconjugated primary antibody, followed by a labeled antibody directed against the first antibody. It is relatively inexpensive, and is more sensitive than the traditional direct technique because several secondary antibodies are likely to react with a number of different epitopes of the primary antibody, thereby amplifying the signal as more enzyme molecules are attached per target site. Horseradish peroxidase is the most commonly used enzyme for indirect antibody enzyme-complex techniques. Fig. 22-2. Immunohistochemistry- Indirect Technique Two-step Indirect Technique In this technique, an unconjugated primary antibody first binds to the antigen. An enzyme-labeled secondary antibody directed against the primary antibody (now the antigen) is then applied, followed by the substrate​- chromogen solution. If the primary antibody is made in rabbit or mouse, the secondary antibody must be directed against rabbit or mouse immunoglobulins, respectively. Cross-reactivity is eliminated by using pre​absorbed secondary antiserum (i.e., antiserum that has been absorbed with immunoglobulins from the species under investigation). Three-step Indirect Technique In this technique, a second enzyme-conjugated antibody is added to the previously described indirect technique. The addition of a third layer of antibody serves to further amplify the signal, since more antibodies are capable of binding to the previously bound secondary reagent. Soluble Enzyme Immune Complex Techniques (Unlabeled Antibody Techniques) These techniques utilize preformed soluble enzyme-anti-enzyme immune complex. The staining sequence involves the use of an unconjugated primary antibody, a secondary antibody, the soluble enzyme-anti-enzyme complex, and the substrate solution. Paraffin Wax Section Immunoperoxidase Technique Paraffin wax section immunoperoxidase technique remains a standard method among most laboratories, especially in combination with frozen section processing for immunofluorescence of renal and skin biopsies. Routinely fixed, paraffin-embedded specimens combine good morphology with localization of various cell and tissue markers. It does not require an expensive fluorescence microscope, it can be adapted as a diagnostic procedure for formalin-fixed, paraffin embedded specimen, and immunolabeling can be correlated with morphology. However, it is more time consuming than frozen-section immunofluorescence. Especially for renal and skin biopsies, proteolytic enzyme digestion is necessary, and is best achieved by using trypsin, chymotrypsin or protease. Non- immune serum is required to block nonspecific staining, especially when polyclonal antibodies are employed. Avidin-biotin labeling, especially with peroxidase, is currently the most popular system used in diagnostic laboratories. The most popular fixative for paraffin embedded sections is neutral buffered formalin. There may be shrinkage or distortion during fixation or subsequent paraffin-embedding, but generally, formalin-based fixatives are excellent for most immunostains. 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 digestion and/or antigen retrieval, particularly if polyclonal antisera are used. Mercuric chloride-based fixatives such as formol sublimate and B5 have gained some popularity because they improve cytological preservation and minimize the distortion associated with formaldehyde-based fixatives. They cause considerable hardening of tissue because of their coagulative properties, allowing thin slices to be made. These types of fixatives are particularly suitable for the demonstration of intracytoplasmic antigens. B5 is widely recommended for the fixation of lymph node biopsies, both to improve the cytological detail and to enhance immunoreactivity with the anti​- immunoglobulin antisera used in phenotyping B cell lymphomas. B5-fixed, paraffin embedded tissue sections show excellent results with cytoplasmic immunoglobulins. However, surface membrane immuno-globulin is not stained as readily. As a general rule, enzyme pretreatments do not improve, and may actually hinder immunostaining of tissue fixed in mercuric chloride-based coagulative fixatives. Alcohol is not widely used as a fixative for routine histological techniques because of its poor penetrating ability. However, small pieces of tissue are fixed rapidly and show good cytological preservation. Since alcohols are coagulant fluids and do not form additive compounds, they permit good antibody penetration and do not block immunoreactive determinants. Alcohol fixation is more commonly used in research laboratories where the size of specimens and handling requirements are different from those observed in routine histopathology. It is also generally applied to frozen sections or smears. Preparing paraffin wax sections for immunostaining 1 Cut 3-S µm sections and place on cleaned glass slides. Vectabond or APES-coated slides may be used to assist with section adhesion. 2. Place sections at 60°C microwave oven overnight (if processing sections without adhesives) or at 56°C for 1 to 2 hours (if Vectabond- or APES​coated slides are used). Heating on a hot plate at high temperature is not recommended because it can be detrimental to the antigen. 3. Deparaffinize sections in xylene and bring to absolute alcohol. 4. Block endogenous peroxidase activity by incubating in 0.5% hydrogen peroxide in methanol for 10 minutes.  5. Rehydrate, wash well in running water and transfer to tris-buffered saline (TBS). 6. To retrieve antigen: a. Preheat slides at 37°C in distilled water. b. Transfer to freshly prepared trypsin solution at 37°C for 15 to 120 minutes (depending on specimen size, duration of fixation and rate of fixation), using 0.1% trypsin in 0.1% calcium chloride in distilled water, adjusted to pH 7.8 using 0.1M NaOH and preheated at 37°C. c. Terminate trypsin enzyme digestion by transferring the sections to cold running tap water. Peroxidase-Antiperoxidase (PAP) Technique The peroxidase-antiperoxidase (PAP) technique is an indirect antibody enzyme-complex technique where the soluble peroxidase-antiperoxidase complex is bound to unconjugated primary antibody (e.g. rabbit anti-human IgG) by a second layer of "bridging" antibody usually a swine anti-rabbit antibody) that then binds to both the primary antibody and the rabbit PAP complex. Fig. 22-3. Peroxidase-Anti-Peroxidase (PAP) Technique Horseradish peroxidase is the most widely used enzyme for labeling. Combining horseradish peroxidase with the most common chromogen, diaminobenzidine (DAB), results in a stable, insoluble dark brown reaction end product when antigen is present in the tissue. To block endogenous peroxidase activity, the sections are pre-incubated in absolute methanol containing hydrogen peroxide. Alkaline phosphatase antibodies raised in mouse, by the same principle, can also be used to form alkaline phosphatase-anti-alkaline phosphatase complexes (APAAP). The major advantage of APAAP procedure compared to the PAP technique is the lack of interference from endogenous peroxidase activity. APAAP technique is recommended for use on blood and bone marrow smears because of the potential distraction of endogenous peroxidase activity on PAP staining. Endogenous alkaline phosphatase activity is usually blocked by adding levamisole to the substrate solution. Peroxidase-Antiperoxidase (PAP) Technique for Paraffin Sections Solutions: Buffer Wash: O.OO5M Tris buffered saline TBS) Tris-Buffered Saline Wash (O.OO5M TBS) Distilled water 1 liter Sodium chloride 8 gm TRIS (hydroxymethyl methylamine) 0.6 gm M HCl 4.4 ml. If necessary, adjust final pH to 7.6 with either 1 M HCl or 0.2 M Tris solution. Substrate: DAB (Diaminobenzidine tetrahydrochloride) Tris-HCL Buffer (recommended for DAB) 0.2 M Tris (containing 24.228 g/l) 12 ml 0.1 M HCl 19 ml. Distilled water 19 ml. DAB Solution DAB 5 mg. Tris-HCI buffer (pH 7.6) 10 ml. H2O2 0.1 ml. (Freshly prepared and added just before use) Method: 1. Bring sections to Tris-buffered saline (TBS). 2. Drain off and wipe around section. 3. Incubate with 1:10 dilution of normal swine serum for 10 minutes. Drain, but do not wash off before applying primary antibody. 4. Incubate in optimally diluted rabbit primary antibody for 30 to 60 minutes. 5. Gently wash in TBS. 6. Incubate in optimally diluted swine anti-rabbit antibody for 30 minutes. 7. Gently wash in TBS. 8. Incubate in optimally diluted rabbit peroxidase anti-peroxidase for 30 minutes. 9. Gently wash in TBS. 10. Incubate in freshly prepared DAB solution at room temperature until  a dark brown reaction product is obtained, usually after 5 to 10 minutes. The reaction end-product resists alcohol dehydration and clearing in xylene. 11. Rinse in TBS and wash in running water. 12. Counterstain in hematoxylin. 13. Dehydrate, clear and mount. 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 such as 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 to 30 minutes at room temperature (20 to 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 to 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 non​- immunological 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. In the context of antibody-mediated antigen detection, nonspecific binding causes high background staining that can mask the detection of the target antigen. Thus, how to block non-specific interactions without reducing the antibody-epitope binding is the challenge to overcome in this case. Sources of unwanted non-specific staining include endogenous enzymes or fluorochromes, endogenous biotin, endogenous antibody binding activity and cross reactivity of the secondary reagents with endogenous proteins. They result in high background causing difficulties in visualizing the antigen of interest in its appropriate cellular location. However, the use of a blocking reagent prior to incubation of the sample with the primary antibody can effectively block non- specific staining interactions. Common blocking buffers include normal serum, non-fat dry milk, BSA or gelatin, and commercial blocking buffers with proprietary formulations are available for greater efficiency. Avidin-Biotin Complex (ABC) Techniques  The Avidin-Biotin Complex technique 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. 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. Alternatively, the enzymes are biotinylated and the avidin-binding sites are occupied by the biotinylated label, forming the avidin-biotin complex. These are supplied commercially as two separate reagents, biotinylated label and avidin or streptavidin, and are added together 30 minutes before use. The basic sequence of staining consists of primary antibody, biotinylated secondary antibody, followed either by the preformed (strept) avidin-biotin- enzyme complex of the avidin-biotin complex (ABC) technique or by the enzyme labeled streptavidin. Formation of ABC complex requires that the solutions of the (strept)avidin and biotinylated enzyme are mixed and prepared at least 30 minutes before use. All incubations are carried out at room temperature. TISSUE Fig. 22-4. Avidin-Biotin Complex (ABC) Technique Method: 1. Paraffin section or frozen section to water and rinse in PBS-Tween 20 twice for 2 minutes each time. 2. Perform antigen retrieval if necessary. 3. Incubate sections in normal serum – species the same as secondary antibody. Note: Since this protocol uses avidin-biotin detection system, avidin/biotin block may be needed based on tissue type. 4. Incubate sections in primary antibody at appropriate dilution for 1  hour at room temperature or overnight. Note: No serum blocking is needed if antibody diluent is used. 5. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 6. Incubate sections in peroxidase blocking solution for 10 minutes at room temperature. Note: For acetone fixed frozen sections, perform this peroxidase blocking step using 0.3% H2O2 in methanol prior to primary antibody incubation to avoid tissue destruction. 7. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 8. Incubate sections in biotinylated secondary antibody in PBS for 30 minutes at room temperature. 9. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 10. Incubate sections in ABC-Peroxidase Solution for 30 minutes at room temperature. 11. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 12. Incubate sections in peroxidase substrate solution. 13. Rinse in PBS-Tween 30 buffer 3 times for 2 minutes each time. 14. Counterstain with counterstain solution. 15. Rinse in running tap water for 2-5 minutes. 16. Dehydrate through 95% ethanol for 1 minute, and then 100% ethanol 2 times for 3 minutes each time. 17. Clear in xylene 2 times for 5 minutes each time. 18. Coverslip. Labeled Streptavidin Biotin Technique (LSAB Procedure) A labeled avidin-biotin (LAB) method has been recently introduced and is found to be 4 to 8 times more sensitive than the old ABC method. Also, avidin has now been largely replaced by the use of streptavidin, leading to the labeled streptavidin-biotin (LSAB) method. The staining sequence consists of primary rabbit (or mouse) antibody, biotinylated anti-rabbit (or anti-mouse) immunoglobulin and streptavidin-enzyme conjugate. The color reaction is then developed with the appropriate substrate/ chromogen, such as horseradish peroxidase. Method: 1. Paraffin section or frozen section to water and rinse in PBS-Tween 20 times for 2 minutes each time. 2. Perform antigen retrieval if necessary. 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 type. 4. Incubate sections with primary antibody at appropriate dilution for 1 hour at room temperature or overnight. No serum blocking is needed if antibody diluent is used. 5. Rinse in PBS-Tween 20 buffer 3 times for 2 minutes each time. 6. Incubate sections in peroxidase blocking solution for 10 minutes at room temperature. Note: For acetone fixed frozen sections, perform this peroxidase blocking step using 0.3% H2O2 in methanol prior to primary antibody incubation to avoid tissue destruction. 7. Rinse with PBS-Tween 20 buffer 3 times for 2 minutes each time. 8. Incubate sections in Biotinylated secondary antibody in PBS for 30 minutes at room temperature. 9. Rinse with PBS-Tween 20 buffer 3 times for 2 minutes each time. 10. Incubate sections in HRP-Streptavidin solution for 30 minutes at room temperature. 11. Rinse with PBS-Tween 20 buffer for 3 times for 2 minutes each time. 12. Incubate sections in peroxidase substrate solution. 13. Rinse with PBS-Tween 20 buffer for 3 times for 2 minutes each time. 14. Counterstain with hematoxylin. 15. Rinse in running tap water for 2-5 minutes. 16. Dehydrate through 95% ethanol for 1 minute, and then 100% ethanol 2 times for 3 minutes each time. 17. Clear in xylene 2 times for 5 minutes each time. 18. Coverslip. Immunofluorescence Method Immunofluorescence technique has become an emerging prime alternative to chromogenic approaches to IHC as it has the ability to generate high- resolution images for protein localization studies and also the capacity to quantitate the fluorescent signal. Immunofluorescent methods are extensively used to detect antibodies, particularly for the diagnosis of glomerular disease in frozen sections of renal biopsies. It is also applied to skin biopsies of patients with systemic lupus and vasculitis, to examine the pattern of deposition of immunoglobulins. In addition, technical advances in microscope development and fluorophore have widened the selection of colors to use for both single- and multi-color fluorescence microscopy greater than ever. Immunofluorescence (IF) method is used in the evaluation of cells in suspension, cultured cells, tissue, beads and microarrays for the detection of specific proteins on both fresh and fixed samples. Its practical application in laboratory include: (a) the analysis of antigens in fresh, frozen or fixed tissues, sub-cellular localization of antigens in tissue culture monolayers and observation of bacterial or parasitic specimens, (b) detection and localization of the presence or absence of specific DNA sequences on chromosomes; and, (c) defining the spatial-temporal patterns of gene expression within cells/tissues. Fluorescence and phosphorescence are both types of luminescence. When molecules with luminescent properties absorb light, they emit light of a different wavelength. In the immunofluorescence method, antibodies are chemically conjugated to fluorescent dyes such as fluorescein isothiocyanate or 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 a flow cytometer, array scanner or automated imaging instrument, or visualized using fluorescence or confocal microscopy. Successful immunofluorescent techniques depend on adequate preservation of substrate antigens, adequacy of antibody conjugate, careful staining and incubation procedures, and quality of the fluorescence microscope. Direct immunofluorescence technique for solid tissue biopsies 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) at 1:10 dilution. In this technique, the tissue is reacted directly with a fluorescein-conjugated antibody specific for the material being sought within the tissue. Solutions: Tris-Buffered Saline Wash (0.005M TBS) Distilled water 1 liter Sodium chloride 8 gm TRIS (hydroxymethyl methylamine) 0.6 gm 1M HCl 4.4 ml If necessary, adjust final pH to 7.6 with either 1 M HCl or 0.2M  Tris solution. Substrate: DAB (Diaminobenzidine tetrahydrochloride) DAB 5 mg Tris-HCl buffer (pH 7.6) 10 ml Horse radish peroxidase 0.1 ml (Freshly prepared and added just before use.) Method: 1. Bring sections to Tris-buffered saline (TBS), drain off, and incubate in non-immune serum. 2. Drain off and wipe around section. 3. Incubate in optimally diluted peroxidase-labeled primary antibody for 1-15 hours (traditional direct method) at ambient temperature or 4°C; or in EPOS peroxidase pre-diluted antibody (DAKO) for 1-2 hours at ambient temperature (enhanced polymer one-step method). 4. Gently wash in TBS. 5. Incubate in freshly prepared DAB solution at room temperature until a dark brown reaction product is obtained, usually after 5 to 10 minutes. The reaction end-product resists alcohol dehydration and clearing in xylene. 6. Rinse in TBS and wash in running water. 7. Dehydrate through 95% ethanol for 1 minute, and then 100% ethanol 2 times for 3 minutes each time. 8. Clear in xylene 2 times for 5 minutes each time. 9. Coverslip. Results: Apple-green fluorescence when fluorescein is used as fluoro-chrome; Orange-red fluorescence with rhodamine conjugates Fig. 22-5. Fluorescein-conjugated antibody against cytoplasmic immunoglobulin in a patient with lymphocytic lymphoma and macroglobulinemia Notes: 1. The slides may be stored at 4°C for one year, but may show decreased fluorescent staining. 2. Avoid direct sunlight on the slides at all times. 3. Cover slip should not be moved or maneuver when mounting, to prevent distortion of the tissue. 4. The tissue sections must be kept moist at all times to prevent artefactual staining. 5. Conjugates prepared from poor quality antibody tend to produce inferior results, often with weak positive results against high levels of background staining. Indirect immunofluorescence technique The indirect immunofluorescence technique is mainly used for the detection of autoantibodies in the patient's serum, including the anti-nuclear antibody (ANA), anti-mitochondrial antibody (AMA), and liver-kidney microsomal antibody. Frozen Section Immunofluorescence Immunocytochemistry can also be employed to identify antigens in fresh frozen sections. Frozen section immunofluorescence is a relatively simple, rapid and sensitive technique that is easily reproducible, particularly among histopathologists who are experienced with fluorescent antibody techniques. However, immunofluorescent labeling requires considerable skill, is not permanent, and often fades within days after the sections have been immunostained. It also requires the use of a costly fluorescence microscope, which can be a major drawback. It is always advisable to also process part of the biopsy for paraffin wax immunohistochemistry in order to visualize and correlate labeling with the morphology of the tissue that is being studied. Cryostat sections give much better antigen preservation than paraffin sections. Additionally, fixative can be used with cryostat sections, so that a different and optimal fixative can be selected for each antigen, all taken from the same block. 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. However, prolonged storage of OCT-embedded frozen tissue will cause gradual loss of cellular antigens, while snap-frozen fresh tissue, without supporting media, can be stored at -70°C for long periods of time without appreciable antigen loss. Acetone is generally used as fixative to preserve the antigen, to destroy harmful infective agents, and to allow a wide range of primary antibodies to be employed without destroying many of the epitopes. 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. Excess buffer is then drained, and the area around the section is wiped dry. 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 1. Snap-freeze fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. 2. Store frozen blocks at -80oC. 3. Cut 4-8 m thick cryostat sections. 4. Mount cryostat sections on either super-frost plus slides or gelatin coated slides. 5. Store slides at – 80oC. 6. Prior to staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 5 minutes. Air dry for 30 minutes. 7. Wash in PBS. IN-SITU HYBRIDIZATION In-situ hybridization shares some similarities with the antigen-antibody reaction that forms the basis for immunohistochemistry. However, it is based on the specificity of the interaction of a probe with the target nucleic acid, rather than the target protein or immunogen. As with immunohistochemistry, optimal results for in-situ hybridization will be achieved only with carefully fixed and processed tissue samples. Formalin​ fixed paraffin-embedded material appears to be the best choice in a diagnostic setting. 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. Because of unavoidable variations in tissue fixation and processing, the use of an endogenous positive control probe is absolutely essential. In an aqueous environment, the thermodynamically stable conformation of a nucleic acid is that of a double-stranded helix. A double-stranded molecule may be separated or denatured into two single strands that disrupt the stabilizing hydrogen bond between complementary bases, i.e., adenine pairs with thymine (or uracil) and cytosine with guanine. Heat and formamide, which both break hydrogen bonds, are the commonly used denaturants. 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". In a hybridization assay, the two sources are the target (sample) and the probe nucleic acids. A probe is simply a known fragment of nucleic acid with a label that can be detected in some fashion. It forms a hybrid molecule with a sample that contains nucleic acids complementary to its sequence. The process of searching a sample for specific nucleic acid sequences is termed "hybridization reaction". Majority of the probes now available are produced either by recombinant nucleic acid technology or through chemical synthesis. 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. 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. The general steps of in​- situ hybridization are basically similar to the steps followed for immunohistochemistry. Detection of radio-labeled probes is usually achieved with autoradiography. Detection of affinity-labeled probes is achieved with methods similar to those previously discussed for immunohistochemistry. Non-isotopic labeling has been initially achieved through the production of biotin-labeled analogue of deoxyuridine triphosphate. Biotin itself cannot generate signals, so that polynucleotides with biotin incorporated into their structure are detected indirectly through high-affinity interaction with avidin or streptavidin chemically linked or complexed to a colorimetric enzyme or fluorescence tags. Alkaline phosphatase systems with tartrazine substrates are reported to yield good results. In most applications, the amount of antigen greatly exceeds the amount of nucleic acid target in tissues, so that low background is essential for optimal results. Fig. 22- 6. In-Situ Hybridization Although numerous well-characterized primary antibodies for immunohistochemistry are now readily available through commercial sources, nucleic acid probes are only beginning to be developed. The lack of commercial reagents is one of the most significant factors limiting the diagnostic application of in-situ hybridization in many laboratories. REFERENCES Adams JC. (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochemistry and Cytochemistry 40: 1457. Battifora H. (1991): Assessment of antigen damage in immunohistochemistry. American Journal of Clinical Pathology 96: 669. Baker JR. (1970) Principles of Biological Microtechnique. Methuen, London. Boenisch, T. (2001) Dako Handbook on lmmunochemical Staining Methods. 3rd ed. DakoCytomation, Carpinteria, California. Bullock GR, Petrusz P, eds. (1982) Techniques in Immunocytochemistry. Academic Press, London, Vol 1. Chan JKC. (2000) Advances in immunohistochemistry. Impact on surgical pathology practice. Sem. Diagn. Pathol. 17(3): 17–77. Chaubert P, Bertholet MM, Correvon M, Laurini S, Bosman FT. (1997) Simultaneous double immunoenzymatic labeling: a new procedure for the histopathologic routine. Mod Path. 10: 585-591.