Evaluation of immunohistochemical staining of human duodenal endocrine cells after microwave antigen retrieval NILS NYHLIN, MAGDY EL-SALHY , OLOF SANDSTRO ¨ M and OLE SUHR Section for Gastroenterology and Hepatology, Department of Medicine, University Hospital, Umea ˚, Sweden Received 28 August 1996 and in revised form 18 November 1996 Summary The effect of microwave antigen retrieval on the immunostaining of human duodenal endocrine cells in formaldehyde- fixed, paraffin-embedded material was investigated. The sections were immunostained by the avidin-biotin complex (ABC) and immunogold-silver autometallography (IGSS) methods with and without prior microwave treatment. Dilutions of up to 1:30 000 of the following antisera antibodies were used: anti-chromogranin A, anti-chromogranin AB, anti-secretin, anti-gastrin, anti-gastric inhibitory polypeptide, anti-somatostatin and anti-serotonin. The detection threshold for all the antibodies was lower after antigen retrieval, and the primary antibody could be used in higher dilutions. The dilutions varied for different antibodies and were between two and ten times the optimal dilution without antigen retrieval. At extremely high dilutions of, or without, the primary antibody, non-specific staining of some lymphocytes and the mucus of some goblet cells was observed when the avidin method was applied, but not with the immunogold technique. This phenomenon was not observed when optimal dilution or a lower dilution was used. This seems to have been caused by the binding of the avidin-biotin complex to epitopes in these structures unmasked by microwave treatment when competition with specific binding sites was absent. Introduction Microwave antigen retrieval (AR) in formaldehyde- fixed paraffin sections was introduced by Shi et al. (1991). This method has been applied since then on a large number of membrane, cytoplasmic and nuclear antigens (Cattoretti et al., 1992; McKee et al., 1993; Suurmeijer & Boon, 1993; Cuevas et al., 1994; Evers & Uylings, 1994; Taylor et al., 1994; von Wasielewskiel et al., 1994; Yachins & Trojanowski, 1994; Hazelbag et al., 1995; Shi et al., 1995). It has been reported that microwave antigen retrieval increases the sensitivity of the immunostaining by lowering the threshold of antigen detection. Thus, antigens that could only be visualized in frozen section can be detected in formaldehyde-fixed paraffin sections. Moreover, anti- gens that could already be demonstrated in routinely processed material can be detected at higher dilu- tions of the primary antibodies. The way in which antigen retrieval affects the formaldehyde-fixed and paraffin-embedded tissue is not yet clear. It has been assumed, however, that the AR acts by breaking the formaldehyde-induced links between epitopes and unrelated proteins, by extrac- tion of diffusible blocking proteins and probably through precipitation of proteins and rehydration of the tissue section, allowing better penetration of the antibody (Suurmeijer & Boon, 1993). To the best of our knowledge, the effect of AR has not been systematically investigated in the endocrine cells of the gut. The aim of the present study was therefore to investigate and evaluate the effect of AR on the sensitivity and specificity of the immunos- taining of the human duodenal endocrine cells. Materials and methods TISSUE SPECIMENS Duodenal biopsy specimens were obtained from four healthy volunteers (two women and two men; mean age 26 years; range 21–28 years), who had given their written consent. These volunteers had no gastrointestinal com- plaints. The investigation was approved by the local committee for medical ethics at the University of Umea ˚. The biopsy specimens were obtained during gastroduode- nal endoscopic examination after overnight fast. They were Histochemical Journal 29, 177–181 (1997) 0018–2214 1997 Chapman & Hall To whom correspondence should be addressed.