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

TL;DR

This study highlights common pitfalls in immunohistochemical detection of insulin-degrading enzyme, emphasizing how certain procedures can lead to false positives or negatives, and provides guidance to improve accuracy in tissue analysis.

Contribution

The paper identifies specific immunohistochemical pitfalls affecting IDE detection and offers optimized protocols to avoid artifacts in human tissue analysis.

Findings

Endogenous peroxidase inhibition can cause false positive IDE staining.

Incubation temperature affects secondary antibody binding and staining accuracy.

Proper blocking and incubation conditions are crucial for reliable immunohistochemistry results.

Abstract

Previously, we have identified the cytoplasmic zinc metalloprotease insulin-degrading enzyme(IDE) in human tissues by an immunohistochemical method involving no antigen retrieval (AR) by pressure cooking to avoid artifacts by endogenous biotin exposure and a detection kit based on the labeled streptavidin biotin (LSAB) method. Thereby, we also employed 3% hydrogen peroxide(H2O2) for the inhibition of endogenous peroxidase activity and incubated the tissue sections with the biotinylated secondary antibody at room temperature (RT). We now add the immunohistochemical details that had led us to this optimized procedure as they also bear a more general relevance when demonstrating intracellular tissue antigens. Our most important result is that endogenous peroxidase inhibition by 0.3% H2O2 coincided with an apparently positive IDE staining in an investigated breast cancer specimen whereas combining a block by 3% H2O2 with an incubation of the biotinylated secondary antibody at RT, yet not at 37 degrees Celsius, revealed this specimen as almost entirely IDE-negative. Our present data caution against three different immunohistochemical pitfalls that might cause falsely positive results and artifacts when using an LSAB- and peroxidase-based detection method: pressure cooking for AR, insufficient quenching of endogenous peroxidases and heating of tissue sections while incubating with biotinylated secondary antibodies.