Comparative study of antigen retrieval heating methods: Microwave, microwave and pressure cooker, autoclave, and steamer

Clive R. Taylor, Shan Rong Shi, Chen Chen, Lillian Young, Christina Yang, Richard J. Cote

Research output: Contribution to journalArticlepeer-review

131 Scopus citations


We present a study comparing the most popular heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HCl buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies; the microwave pressure cooker, extended microwave heating (5 min x 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min x 2). Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AIR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for 'maximal retrieval' as a common end point for all laboratories.

Original languageEnglish
Pages (from-to)263-270
Number of pages8
JournalBiotechnic and Histochemistry
Issue number5
StatePublished - Sep 1996


  • antibody
  • antigen retrieval
  • autoclave
  • formalin fixation
  • immunohistochemistry
  • paraffin sections
  • pressure cooker


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