Immunohistochemical staining is a valuable tool for detecting specific antigens in tissues. In order to perform the standard staining procedure, first the tissue section has to be deparaffinized and then rehydrated before applying the primary antibody. Enzyme-conjugated secondary antibodies are then applied and the specific staining can be visualized after adding the enzyme-specific substrate. Occasionally, when weak or no staining is observed, an antigen "unmasking" by enzyme digestion, may be required.
The following procedure describes the application of peroxidase or alkaline phosphatase conjugates in the immunohistochemical labeling of formalin-fixed, paraffin-embedded tissue sections.
Figure 1.Immunohistochemical staining
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Deparaffinization and Rehydration | Antigen Retrieval - Unmasking | Inactivation of Endogenous Peroxidase
Note: This step is performed only in cases where weak or no staining occurs, or for antigens requiring "unmasking" according to the primary antibody specifications.
There are several possible ways for retrieval depending on the antibody and the antigen:
Note: This step is performed only when using peroxidase-conjugated secondary antibodies or ExtrAvidin-Peroxidase.
a. Pre-incubation of the sample with 5% BSA for 10 min. prior to the primary antibody reaction may decrease background staining. For best results with animal tissues, use 5 to 10% normal serum from the same species as the host of the secondary antibody.
b. Optimal dilution and incubation times should be determined for each primary antibody prior to use.
Option 1 - Biotin/ExtrAvidin Detection
Option 2 - Enzyme-Labeled Secondary Antibody
It is recommended to prepare the substrate mixture during the final wash step.
Note: Addition of 1 mM levamisole (Product No. L9756) to the alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)
Note: When using AEC substrate, do not use alcohol-containing solutions for counter-staining (e.g., Harris' hematoxylin, acid alcohol), since the AEC stain formed by this method is soluble in organic solvents. The slide must not be dehydrated, brought back to toluene (or xylene), or mounted in toluene-containing mountants.
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