Antigen retrieval techniques in immunohistochemistry: comparison of different methods

TLDR

The authors compared three heat‑based antigen retrieval fluids—citrate (pH 6.0), Tris–HCl (pH 8.0), and EDTA–NaOH (pH 8.0)—applied by microwave or pressure cooking on formalin‑fixed sections, using APAAP and SABC detection, and also evaluated protease digestion and no retrieval. All but one antibody benefited from retrieval, protease digestion was the gold standard for five antibodies, while 55 antibodies performed optimally with heat‑based retrieval, with EDTA yielding the strongest staining and most marked cells, and these results were consistent across tissues, methods, and heating devices, with pressure cooking offering a practical time saving of 1 min 30 s per batch. © 1997 by John Wiley & Sons, Ltd.

Abstract

Routine sections of normal and pathological samples fixed in 10 per cent buffered formalin or B5, including EDTA-decalcified bone-marrow biopsies, were tested with 61 antibodies following heating in three different fluids: 0·01 m citrate buffer (pH 6·0), 0·1 m Tris–HCl (pH 8·0), and 1 mm EDTA–NaOH solution (pH 8·0). The sections underwent either three cycles of microwave treatment (5 min each) or pressure cooking for 1–2 min. The alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique was used as the standard detection method; with 16 antibodies a slightly modified streptavidin–biotin complex (SABC)-immunoperoxidase technique was applied in parallel. The results obtained were compared with those observed without any antigen retrieval (AR), or following section digestion with 0·05 per cent protease XIV at 37°C for 5 min. Chess-board titration tests showed that all antibodies but one profited by AR. Protease XIV digestion represented the gold standard for five antibodies, while 55 produced optimal results following the application of heat-based AR. By comparison with the other fluids, EDTA appeared to be superior in terms of both staining intensity and the number of marked cells. These results were independent of tissue processing, immunohistochemical approach, and heating device. Pressure cooking was found to be more convenient on practical grounds, as it allowed the simultaneous handling of a large number of slides and a time saving of 1 min 30 s, representing the proper time for the treatment. © 1997 by John Wiley & Sons, Ltd.