A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections

Abstract

The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical 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 μm free floating sections, slide-mounted cryostat sections and paraffin-embedded 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.

Introduction

The masking of antigens by aldehyde fixation is a well-known problem in immunohistochemistry. Different antigen retrieval methods have been developed to overcome this problem, including enzymatic predigestion 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 microwave 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 (Suurmeijer and Boon, 1993). The microwave-heating antigen retrieval approach has come to be widely accepted as the most sensitive and reliable among the heating methods 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, 1997, Newman and Gentleman, 1997). The microwave heating method is effective for formalin-fixed tissue and for paraffin-embedded tissue. It also improves immunohistochemical labeling in frozen tissue (De Hart et al., 1996). Typically the tissue is immersed in a sodium citrate solution during the heating process.

The microwave heating method is not, however, without its drawbacks. For instance, it is difficult to produce a controlled and uniform heating temperature with a microwave oven. Additionally, microwave heating does not work well for free-floating sections because the high temperatures and vigorous bubbling that can occur damage the sections, and even expel them from the solution. For example, while some authors have reported some success with immunolabeling of 40 μm thick free floating vibratome sections that had been microwave heated for antigen retrieval (Shiurba et al., 1998), the sections did show wrinkling. To address such problems, Evers and Uylings (1994a) recommended that tissue first be cut into 0.5 cm thick slices, microwave heated to retrieve the antigens, and then sectioned into 95 μm or thinner sections prior to immunostaining. This procedure, however, has the drawback that it involves two sectioning steps.

The effectiveness of various heating methods for antigen retrieval suggests that heating is the key variable in producing a successful immunolabeling outcome (Suurmeijer and Boon, 1993, Bankfalvi et al., 1994, Eagle et al., 1997). Other major factors that appear to influence the effectiveness of antigen retrieval include the pH of the solution in which the tissue is immersed during antigen retrieval, the concentration of this solution, the heating temperature, and the duration of the heating time (Evers and Uylings, 1994b, Shi et al., 1995). With these points and the relative uncontrollability of the temperature with microwave heating in mind, we examined the effectiveness of sub-boiling water-bath heating for antigen retrieval in free floating and slide-mounted sections. This approach, in principle, provides considerably more precise and uniform temperature control than does microwave heating. We also systematically explored different pH values, different temperatures, different heating times, and different sodium citrate solution concentrations to determine the optimal parameters for antigen retrieval with water-bath heating.