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Reagents can be applied manually by pipette, or this protocol can be adapted for automated and semi-automated systems if these are available.
Carry out incubations in a humidified chamber to avoid tissue drying out, which will lead to non-specific binding and high background staining. A shallow plastic box with a sealed lid and wet tissue paper at the bottom is an adequate chamber. Keep slides off the paper and lay flat so that the reagents don't drain off.
?Cut a plastic serological pipette into lengths to fit your incubation chamber then glue them in pairs to the bottom of the chamber, with each pair about 4 cm apart. This provides a level and raised surface for the slides to rest on, away from the wet tissue paper.
Dilutions of the primary and secondary antibody are listed on the datasheets or are determined by testing a range of dilutions. Adjust dilutions appropriately from the results obtained.
For enzymatic methods, horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most commonly used enzymes. There are a number of chromogens are used with these enzymes.
Perform antigen retrieval before commencing with immunostaining if necessary.
For enzymatic detection (HRP or AP secondary conjugates)
For fluorescent detection
Materials and reagents
Peroxidase block (40 mL)
To achieve a stronger signal, various strategies have been developed to add more enzyme or fluorophore to the target of interest.
Avidin-biotin complex (ABC)
This technique uses the high affinity of avidin for biotin, an enzyme co-factor in carboxylation reactions. Avidin has four binding sites for biotin and binding is essentially irreversible.
In brief, the primary antibody is bound to the protein of interest. A biotinylated secondary antibody is then bound to the primary antibody. In a separate reaction, a complex of avidin and biotinylated enzyme is formed by mixing the two in a ratio that leaves some of the binding sites on avidin unoccupied. This complex is then incubated with the tissue section after the antibody incubations. The unoccupied biotin-binding sites on the complex bind to the biotinylated secondary antibody. This attaches more enzyme to the target than is possible using an enzyme-conjugated secondary or primary antibody.
The components of the avidin-biotin complex are commercially available in kits and the complex can be used with our biotinylated antibodies. The presence of endogenous biotin in tissues can bind the avidin-biotin complex and cause background staining. Such tissues include kidney, liver, brain, prostate, colon, intestines, and testes.
Labeled streptavidin biotin (LSAB)
This method is similar to ABC as it uses the interaction of streptavidin (similar to avidin in binding affinity) and biotin. The primary antibody is followed by a biotinylated anti-lg secondary antibody, followed by streptavidin conjugated to an enzyme or fluorophore. Streptavidin produces less non-specific background staining than avidin since it is non-glycosylated (unlike avidin) and so doesn’t interact with lectins or other carbohydrate-binding proteins. For a comparison to ABS in which LSAB was shown to be 4–8 times more sensitive (see Giorno R (1984), Diagn Immunol. 2:161–6). See our LSAB ABC kits.
??Both avidin-biotin methods (ABC and LSAB) are losing favor to new polymer-enzyme-antibody products that consist of a secondary antibody (eg anti-mouse and/or rabbit IgG) attached to a polymer-enzyme complex. This is partly because the issue of endogenous biotin is avoided.
?Micro-polymer detection kits for IHC use small linker-based detection modules that can penetrate tissues better than the large complexes used in conventional polymer-based detection, resulting in higher sensitivity.
Tyramide signal enhancing (TSE)
One of the most effective amplification procedures is the patented and licensed method, TSE (also known as TSA or CSA). It is useful for detection of sparse antigens that other systems have difficulty detecting, and for improving results obtained with poorly-performing antibodies.
The method relies on a peroxidase-catalyzed reaction to covalently attach the tyramide portion of tyramine-protein conjugates to the area around the protein of interest, after applying a primary antibody and secondary-HRP conjugate. The covalently attached protein cannot be washed off, even if the slides are treated to remove the antibodies, since the tyramide bond is covalent. A signal is obtained by directing antibody-enzyme or fluorophore conjugate against the protein portion of the tyramine-protein conjugate.
Protocols are provided by Abcam “AS-IS” based on experimentation in Abcam’s labs using Abcam’s reagents and products; your results from using protocols outside of these conditions may vary.??