Journal of Neuroscience Methods 93 (1999) 149 – 162 A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections Yun Jiao, Zhiqiang Sun, Teffy Lee, Francesca R. Fusco, Toya D. Kimble, Christopher A. Meade, Sherry Cuthbertson, Anton Reiner * Department of Anatomy and Neurobiology, College of Medicine, The Uniersity of Tennessee – Memphis, The Health Science Center, 855 Monroe Aenue, Memphis, TN 38163, USA Received 22 March 1999; received in revised form 31 August 1999; accepted 1 September 1999 Abstract The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohisto- chemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10–50 mM sodium citrate solution (pH 8.5–9.0) preheated to and maintained at 80°C in a water-bath, followed by 30 min incubation in 0.3–3% nonfat dry milk to reduce nonspecfic staining. This method is highly effective for both 40 m free floating sections, slide-mounted cryostat sections and paraffin-embed- ded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Immunohistochemistry; Antigen retrieval; Free-floating sections; Paraffin-embedded sections; Water-bath heating www.elsevier.com/locate/jneumeth 1. Introduction The masking of antigens by aldehyde fixation is a well-known problem in immunohistochemistry. Differ- ent antigen retrieval methods have been developed to overcome this problem, including enzymatic prediges- tion of tissue (Battifora and Kopinski, 1986), formic acid pretreatment of tissue (Kitamoto et al., 1987), heat-induced epitope retrieval (Shi et al., 1991), and ultra-sound treatment of tissue (Podkletnova and Alho, 1993). The mechanism underlying successful retrieval of antigens presumably involves breaking formaldehyde- induced crosslinks between the target antigen and other proteins, and/or renaturing the antigen to expose the target epitopes of the antisera. Among these different antigen retrieval methods, heating is the most widely used approach. Various methods have been used to heat tissue, including mi- crowave oven heating (Shi et al., 1991; Cattoretti et al., 1993), autoclave heating (Shin et al., 1991; Bankfalvi et al., 1994; Navabi et al., 1994), pressure cooker heating (Igarashi et al., 1994; Norton et al., 1994; Baiaton et al., 1996; Eagle et al., 1997), and steamer heating (Suurmei- jer and Boon, 1993). The microwave-heating antigen retrieval approach has come to be widely accepted as the most sensitive and reliable among the heating meth- ods since first introduced by Shi et al. (1991). Slight modifications in the original protocol have since been suggested by various authors (Cattoretti et al., 1993; Sherriff et al., 1994; Shi et al., 1996; Evers and Uylings, * Corresponding author. Tel.: +1-901-448-8298; fax: +1-901-448- 7193. E-mail address: areiner@utmem.edu (A. Reiner) 0165-0270/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII:S0165-0270(99)00142-9