An Evaluation of Antigen Retrieval Procedures for Immunoelectron Microscopic Classification of Amyloid Deposits

Abstract

The advantages of using immunoelectron microscopy in amyloid research and surgical pathology for the classification of amyloid deposits are well documented. The aim of this study was to improve single-labeling postembedding immunostaining by testing different antigen retrieval (AR) techniques. Etching and AR procedures were applied to sections from aldehyde-fixed and Epon-embedded autopsy specimens of patients who had suffered from generalized AA amyloidosis, systemic senile ATTR amyloidosis, or generalized κ-light chain amyloidosis. The procedures used were no AR, H2O2, saturated aqueous sodium metaperiodate (mPJ), heating in deionized water (dH2O), heating in sodium citrate buffer (SCB), heating in EDTA (each 91C, 30 min), and combinations of etching and heating. Little effect was evident after treatment with H2O2, mPJ, and heating in dH2O, but the signal density markedly increased after heating in 1 mM EDTA. Heating in SCB affected immunolabeling with anti-transthyretin and anti-κ-light chain, whereas no effect was achieved for immunolabeling with anti-AA amyloid. We concluded that AR may significantly improve immunostaining of specimens that have undergone conventional fixation and embedding procedures for electron microscopy. The effect of AR on the detection of amyloid fibril proteins was probably mediated in part through chelation or binding of metal ions by the AR medium.