Immunohistochemical Detection of Ki67 in Glycol Methacrylate Sections Using Unfixed Tissues and Alkaline Antigen Retrieval Techniques

Abstract

Assessing cell proliferation through immunohistochemical procedures is an important component of many research projects. Unfortunately, published protocols often yield inconsistent results in individual laboratories. We present a method for glycol methacrylate embedding and antigen retrieval with alkaline solutions to detect Ki67 antigen. In brief, fresh tissues are dehydrated through a series of catalyzed resin solutions, and then embedded for standard microtomy at 1.5–2 μm. Cut sections were mounted on slides and pretreated with a 0.2% NaOH solution for 1.5–2 minutes and then stained to detect Ki67 according to standard immunohistochemical protocols. Secondary detection reagents tested in the development of this protocol included HRP/DAB combinations, Au/Ag staining procedures, and fluorescent secondary antibodies. Antigen sites could not be demonstrated in aldehyde-fixed tissues, but were readily demonstrated in the unfixed tissues after alkaline antigen retrieval. The method described is a simple, inexpensive, and rapid procedure to allow antigen detection. The method may have additional utility in the demonstration of other nuclear antigens. (The J Histotechnol 32(2):60 – 63, 2009)Submitted January 18, 2009; accepted with revisions March 25, 2009