Alteration of immunoreactivity by hydrated autoclaving, microwave treatment, and simple heating of paraffin‐embedded tissue sections

Abstract

The effects of treatment in a hydrated autoclave (121 °C, 2 atm for 20 min), microwave oven (in water), and simple heating (60 °C overnight in distilled water or 90 °C for 10 min in ZnSO4) on the stainability of 56 antigens by commercially available antibodies in formalin‐fixed paraffin‐embedded tissue sections were evaluated. The detectability of nuclear antigens, glycoprotein, lymphocytic surface markers, and chromogranin A was significantly and reproducibly improved by these treatments, whereas the detectability of viral antigens and peptide hormones was attenuated or unchanged. This enhancement includes not only the distinctiveness of the positive staining, but also the number of positive cells, as revealed by comparing serial sections. Among these four heating procedures, microwave heating and autoclaving were more effective than the others on p53, c‐erbB‐2, and CA125, whereas simple heating was best for smooth‐muscle actin (HHF35 and CGA7). Generally the effects of the heating procedures for these antigens were consistent among the cases, but the effects on GFAP varied with the case. The alterations we observed could significantly influence the interpretation of immunohistochemical staining of currently popular tumor markers such as p53 in terms of their prevalence (28%vs 64% in gastric cancer; 36%vs 82% in metastatic liver cancer) and other diagnostically important markers.

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

“…The fact that high-temperature heating is the most important factor for retrieval of antigens masked by formalin fixation was demonstrated by Shi et al (1991) and in many subsequent applications of AR-IHC in pathology Igarashi et al 1994;Kawai et al 1994a;Lucassen et al 1993;Suurmeijer and Boon 1993a;Malmstrom et al 1992). Higher temperature in general yields better results of AR-IHC.…”

“…For example, Kawai et al (1994b) found that heating at 90C for 10 min was more effective than heating at 60C for 120 min. Different heating methods have been used for AR-IHC, such as autoclaving (Pons et al 1995;Bankfalvi et al 1994;Igarashi et al 1994;Umemura et al 1994;Shin et al 1991), pressure cooking (Miller and Estran 1995;Norton et al 1994), water bath (Kawai et al 1994b), microwaving (MW) plus plastic pressure cooking Pertschuk et al 1994), and steam heating (Pasha et al 1995;Taylor et al 1995). Again, the temperature achieved by these methods appears to be the critical variable.…”

“…This is a critical issue in interpreting the results of AR-IHC. Thus far, most studies have reported satisfactory results, without false positivity, for a variety of antibodies tested (Brown and Chirala 1995;Lavieille et al 1995;Cuevas et al 1994;Igarashi et al 1994;Taylor et al 1994a;Tenaud et al 1994;von Wasielewski et al 1994;Cattoretti et al 1993;Dookhan et al 1993;Gown et al 1993a;Leong and Milios 1993;Shi et al 1991). Most recently, Baas et al (1996) questioned whether a potential false-positive result of AR-IHC using the MAb DO7 might be obtained after extreme AR treatment.…”

“…A simple method of either heating the routinely fixed paraffin-embedded tissue sections in water or immersing the routinely formalin-fixed, aciddecalcified, celloidin-embedded tissue sections in an NaOH-methanol solution yielded dramatic retrieval results (Shi et al 1991(Shi et al ,1992a(Shi et al ,b,1993a. The AR technique has been used increasingly and has demonstrated its value in many studies for over 200 antibodies Werner et al 1996;Boon and Kok 1995;Brown and Chirala 1995;Cattoretti and Suumeijer 1995;Shi et al 1995b;Cuevas et al 1994;Gu 1994;Gu et al 1994a,b;Igarashi et al 1994;Kawai et al 1994a;Swanson 1994;Taylor and Cote 1994;Cattoretti et al 1993;Gown et al 1993a;Leong and Milios 1993;McKee et al 1993;Norton 1993;Suurmeijer and Boon 1993a,b;Shi et al 1991).…”

Initial Intermittent Microwave Irradiation for Fluorescence In Situ Hybridization Analysis in Paraffin-Embedded Tissue Sections of Gastrointestinal Neoplasia

“…In particular, gastrointestinal tumor specimens are notoriously difficult to handle using standard FISH and conventional cytogenetic techniques because of frequent necrosis, contamination with inflammatory and normal stromal cells, and the poor attachment of the tumor cells (Kitayama et al, 1995). Present techniques involving the use of microwave ovens for antigen retrieval (Igarashi et al, 1994;Taylor et al, 1994) in formalin-fixed, paraffinembedded tissues have led to the suggestion that FISH signals might be enhanced if an appropriate heat treatment is included during sample preparation. To avoid the above-mentioned problems associated with paraffin sections, the FISH protocol was thus modified to incorporate an intermittent, short-term microwave (MW) treatment during the initial period of hybridization.…”