Staining Methods and Detection Systems: Direct/Indirect, Polymers, Amplification, Chromogens, and Multiplexing
Detection architectures
Direct methods label the primary antibody; indirect methods use an unlabeled primary followed by a labeled secondary, gaining signal amplification. Modern routine IHC is usually a two-step polymer: a secondary antibody or compact polymer bearing horseradish peroxidase (HRP) or alkaline phosphatase (AP) and a small-molecule antibody. Avidin-biotin complex (ABC) methods remain in use but are vulnerable to endogenous biotin, especially in liver and kidney.
Amplification
Tyramide signal amplification (TSA/CSA) deposits biotinylated or fluorophore tyramide near the HRP site, giving large gains in sensitivity for low-abundance targets. Amplification also amplifies background and can convert weak nonspecific binding into apparent positivity, so it must be validated with careful negative controls. TSA deposition is also the backbone of many multiplex protocols, since the primary/secondary pairs can be stripped by heating between rounds.
Chromogens and enzymes
HRP with DAB gives a brown, alcohol-insoluble product compatible with hematoxylin and permanent mounting. AEC gives red but is alcohol-soluble, so it must be mounted in aqueous medium and is unsuitable for procedures requiring dehydration. AP with Fast Red or BCIP/NBT gives red or blue-black products, and levamisole is added to block endogenous AP. Endogenous peroxidase is blocked with hydrogen peroxide; endogenous biotin requires a dedicated blocking step.
Multiplexing
Sequential chromogenic or fluorescent multiplexing requires stripping or elution between rounds, species-specific secondaries, or TSA-based workflows. Watch for cross-reactivity, incomplete stripping, and epitope loss during repeated retrieval.
Traps
Brown pigment is not automatically DAB - melanin, hemosiderin, and lipofuscin all mimic it. Do not dehydate an AEC slide through alcohols. Over-amplification is a common source of false positivity in questions.
Rule of thumb
Match chromogen to mounting: DAB for permanent resinous mounting, AEC or AP red for aqueous mounting and for distinguishing two colors in double stains.