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Better IHC Step 3: Detection


Previous: Step 2, Antibody Dilution

Detection reagents carry enzymes to the site of the specific epitope by binding to the primary antibody, either directly or indirectly, through a secondary antibody intermediate. When a chromogenic substrate for horseradish peroxidase (HRP) is introduced, a precipitate forms that deposits at the site of the primary antibody antigen binding event, making it visible upon microscopic examination.

Historically, this interaction is facilitated by biotin and streptavidin. The secondary antibody is conjugated to biotin, which can bind streptavidin, and the streptavidin molecule itself is conjugated to HRP.

This method has several limitations. (Watch to find out what they are.)

Next: Step 4, Chromogen