Tissue Preparation for Histochemistry: Fixation, Embedding, and Antigen Retrieval for Light Microscopy

Abstract

A number of techniques have been developed to use chemical, immunological, and molecular biology assays in histological material. Collectively termed histochemistry, these techniques have allowed us to better understand tissue and organ biology in situ. Success with each of these methods is dependent on the adequate preparation of material. In this article, we describe the basic steps required to prepare tissue for routine histochemical analysis.Histochemical techniques routinely use frozen and paraffin-embedded tissue as a basis for cellular and morphological analysis. Freezing tissue results in less alteration to epitopes and therefore may offer improved staining characteristics compared to techniques based on paraffin embedding. As in conventional histology, the use of fixation and embedding in more rigid media such as wax offers a number of potential advantages related to improved structural detail. Improvements in morphology may however be offset by a loss of antigens. The careful application of antigen retrieval techniques may overcome these deficiencies.