Introduction: To ensure quality control and in compliance with the appropriate quality assurance programs, it is necessary to have detailed Standard Operating Procedures (SOP) The addition of transparent reagent helps paraffin absorb into the tissue. While the direct method is simple, rapid and highly-specific, it has low sensitivity and a limited range of primary antibodies that are directly labeled. 0000003852 00000 n Immunohistochemistry (IHC) is a method for detecting antigens or haptens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. After dehydration, the tissue of interest requires a transparentizing step because the dehydrating agent used in the previous step is immiscible with the paraffin from one of the subsequent steps. Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm. 0000128780 00000 n Blocking should be done at room temperature for 10-30 min (avoid excessive blocking). Differentiation refers to the process of using reagents (e.g. After running nucleolar staining (in aluminum hematoxylin) and differentiation (in HCl and alcohol), the tissue section is transferred from an acid solution to an alkaline solution (e.g. 0000008527 00000 n Before mounting, the sample should be treated with dimethyl benzene, transparent and dehydrated for long-term storage sections.
The antibody-antigen interaction is visualized using either chromogenic detection with a colored enzyme substrate, or fluorescent detection with a fluorescent dye . Formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. Sample collection and preparation play an important role in IHC as the antigen exhibition and location are largely depend on the quality of tissue sample.
0000002837 00000 n An enzyme label is reacted with a substrate to yield an intensely colored product that can be analyzed.
When either the horseradish peroxidase (HRP) or alkaline-phosphatase (AP) system is applied for IHC, activation of endogenous enzymes should be blocked or inhibited to avoid producing non-specific binding.
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The enzymatic IHC technique was introduced by Nakane and Pierce in 1967. 0000002280 00000 n 0000003147 00000 n Protocols, optimization tips, troubleshooting guides, and more for IHC. Similar to DAB, hematoxylin, methyl green and methyl blue are some of the suitable counterstains for AEC. Since xylene has a span and fast contractility to tissue, the tissue should not be immersed for an extended time period or it will be over crisp and too hard.
Coons and co-workers developed the IF technique in 1941.
It helps keep the cross-linking between tissues and maintain antigen. Note: Microscopic slide for IHC is required to be 5 μm. 0000005007 00000 n skin tissue) and dense fibrous tissue are also easier to be sectioned after immersing in cedar oil. 34 0 obj<> endobj The method selected should include consideration of parameters such as the specimen types and assay sensitivity. Microwaving for double indirect …
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Differentiation and bluing should be carried out after staining with Harris Hematoxylin. 0000035889 00000 n Both fresh and fixed tissues can be processed as frozen tissues. The secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. Primary antibody binds specifically to target antigen, Secondary antibody labeled with fluorophore binds to primary antibody, Affix the sample on glass slide (To ensure the validity of fluorescence staining, positive, negative and sample autofluorescence controls should be carried out to confirm there is no non-specific binding.
While many of these stains show specificity for discrete antigens or cellular compartments, other stains will deliver the staining of a whole cell. Tissue samples are typically taken from specimens of various sources: biopsy, surgery, animal model and autopsy. IHC-Paraffin or plastic) or frozen blocks (e. g. IHC-Frozen), and the sections are then probed with primary antibodies against the antigens of interest. After cooling is completed, the tissue will be ready for sectioning and suitable for storage. Due to minimal sclerification created by cedar oil, it is an appropriate transparent agent for fine and soft tissues. First of all, only a relatively small number of standard conjugated (labeled) secondary antibody is needed to be generated for the indirect method.
0000118851 00000 n Pepsin and bromelin are used for retrieving antigens in intercellular substance. Download troubleshootingnhandbooks for IHC, Western blot and ELISA for FREE. Three methods are described here: immunofluorescence (IF), Enzymatic and Affinity. h��mo�6��S�q����w���^c�j4:, The HIER method can be implemented by microwave, high pressure or water bath. In principle, the time is directly proportional to the tissue thickness but inversely proportional to the solution concentration. 0000099136 00000 n Acetone is often used for frozen tissue and cytological smears because it has a span penetrability and dehydration property.
Avidin-Biotin Peroxidase Complex (ABC) and Labeled Streptavidin Binding (LSB) are the two most widely used affinity methods for amplifying the target antigen signal. Beside avidin, there are other methods which involve streptavidin which is a tetrameric biotin-binding protein that is isolated from Streptomyces avidinii. 0000002567 00000 n
However, the slide for nervous tissue should be 20-100 μm to enhance the tracking of never fiber direction. With low toxicity and degradable chemical agent, this solution has gained a broad popularity in IHC, regular pathological examinations and molecular pathology detections due to the use of non-protein cross linking, span DNA/RNA preservation, and absence of cell vacuole, tissue shrinkage and pyknosis. Use enough fixing solution and wash it off completely after fixation, Use tissues of size less than 2 cm × 1.5 cm × 0.3 cm (Thickness < 0.3cm), Dilute 3-Amino Propyl Tri-ethoxy Silane (APES) by acetone, Apply tissue on the coated slide followed by adding a drop of mounting medium (Glycerol Gelatin) on the tissue, Hold the coverslip at 45°, allowing the drop to spread along the edge of the slip, Slowly cover the tissue entirely with the coverslip, Incubate the slide at 80℃ for 1 hour if there is an adhering problem with a tissue section, Place settled coverslip in culture bottle or perforated plate, Take out coverslip after cell growth has reached 60%, Fix coverslip with cold acetone or 4% paraformaldehyde for 10 to 30min, Collect non-adherent cells and wash 2X by cold PBS buffer, Add 30-50 µL to settled slide and smear it evenly, Air dry slide a little bit and cover cell with 4% paraformaldehyde for 2-4 hours, Incubate the paraffin embedded section in 3% H2O2 for 10 min, Incubate the frozen section or cell section in solution composed of methanol and 3% H2O2 (v/v: 4:1) for 30 min, Incubate the sample section in 0.1 mM Levamisole, Note: Levamisole cannot inhibit the AP activation of endogenous intestine tissue, Fix coverslip with cold acetone or 4% paraformaldehyde for 10 to 30 min, Place the sample section into a microwaveable vessel where antigen retrieval reagent is present, Apply microwave radiation to the sample for 5-20 min, Place the sample section into an appropriate vessel where antigen retrieval reagent is present, Place the vessel inside a pressure cooker, Turn on the cooker and heat the sample until it boils, Once boiling starts, turn off the cooker after the sample is allowed to reach full pressure for 1-4 min, Place the vessel and thermometer inside a water bath chamber, Remove the sample from the chamber after it is heated at 92℃ for 20-40 min. Animal’s autoantibody in the serum can bind to the sites in advance. After staining the target antigen by IHC, a secondary stain is usually applied to provide contrast that helps the primary stain more distinct.