an enzyme or a fluorophore, respectively. With chromogenic reporters, an enzyme label reacts with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound. Reaction with DAB can be enhanced using nickel, producing a deep purple/black staining. Fluorescent reporters are small, organic molecules used for IHC detection and traditionally include FITC, TRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization. Target antigen detection methods The direct method is a one-step staining method and involves a labeled antibody (e.g. FITC-conjugated antiserum) reacting directly with the antigen in tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, in contrast to indirect approaches. However, this strategy is used less frequently than its multi-phase counterpart. The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody. As mentioned above, the secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter. Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin- or NeutrAvidin protein-bound enzyme. The difference between these three biotin-binding proteins is their individual binding affinity to endogenous tissue targets leading to nonspecific binding and high background; the ranking of these proteins based on their nonspecific binding affinities, from highest to lowest, is: 1) avidin, 2) streptavidin