What is the correct sequence of steps in the ABC method for immunohistochemistry?

• A. Incubation of primary antibody with tissue sample to allow binding to target antigen; Incubation of biotinylated secondary antibody with tissue sample to allow binding to primary antibody; Pre-incubation of biotinylated enzyme with free avidin to form large ABC complexes; Incubation of the above pre-incubated solution to tissue sample
• B. Incubation of primary antibody with tissue sample; Pre-incubation of enzyme with avidin; Incubation of tissue with the pre-formed ABC complex; Color development
• C. Incubation of tissue with primary antibody; Incubation with enzyme conjugate; Addition of avidin; Chromogen development
• D. Incubation of tissue with primary antibody; Incubation of a biotinylated secondary antibody; Direct application of enzyme without ABC; Incubation and color development

Answer: (A)

In the ABC method, signal amplification comes from the avidin–biotin–enzyme complex bringing multiple enzyme molecules to the antigen site. The correct sequence starts with the primary antibody binding its target antigen in the tissue. Next, a biotinylated secondary antibody binds to that primary antibody, placing biotin at the antigen location. Then the enzyme is pre-bound to avidin to form the ABC complex, which is subsequently applied to the tissue. This final incubation lets the ABC complex attach specifically at the biotin sites created by the secondary antibody, localizing the enzyme exactly where the antigen is. Afterward, chromogen development reveals the signal.

This order is essential because without the biotinylated secondary antibody, there’s no biotin marker for the ABC complex to bind to; without pre-forming the ABC complex (as described), the enzyme wouldn’t be efficiently anchored at the antigen site. Other sequences either omit the biotinylated secondary antibody, apply the enzyme directly without the ABC amplification, or misplace the ABC components, resulting in weaker or absent staining.