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Previously, we have identified the cytoplasmic zinc metalloproteaseinsulin-degrading enzyme(IDE) in human tissues by an immunohistochemical methodinvolving no antigen retrieval (AR) by pressure cooking to avoid artifacts byendogenous biotin exposure and a detection kit based on the labeledstreptavidin biotin (LSAB) method. Thereby, we also employed 3% hydrogenperoxide(H2O2) for the inhibition of endogenous peroxidase activity andincubated the tissue sections with the biotinylated secondary antibody at roomtemperature (RT). We now add the immunohistochemical details that had led us tothis optimized procedure as they also bear a more general relevance whendemonstrating intracellular tissue antigens. Our most important result is thatendogenous peroxidase inhibition by 0.3% H2O2 coincided with an apparentlypositive IDE staining in an investigated breast cancer specimen whereascombining a block by 3% H2O2 with an incubation of the biotinylated secondaryantibody at RT, yet not at 37 degrees Celsius, revealed this specimen as almostentirely IDE-negative. Our present data caution against three differentimmunohistochemical pitfalls that might cause falsely positive results andartifacts when using an LSAB- and peroxidase-based detection method: pressurecooking for AR, insufficient quenching of endogenous peroxidases and heating oftissue sections while incubating with biotinylated secondary antibodies.

Immunohistochemical pitfalls in the demonstration of insulin-degrading enzyme in normal and neoplastic human tissues