AbstractA recently described antigen retrieval method with microwave heating of tissue sections was combined with trypsin digestion to increase the immunostaining intensity of estrogen and progesterone receptors. We used 2 new monoclonal antibodies, the estrogen receptor (ER)-specific ER/D5 and the progesterone receptor (PR)-specific PRlOA9, to detect hormone receptors in tissue sections of routinely fixed and embedded breast cancer specimens. These 2 antibodies react with their antigens only following microwave pretreatment of the fixed-embedded sections. When we used trypsin digestion before the microwave heating, the intensity of nuclear hormone receptor immunostaining increased. In some cases, we also found cytoplasmic and/or membrane-associated staining when using the PR-specific PRlOA9 antibody. However, when we used trypsin digestion following the microwave heating, the nuclear staining intensity of ER decreased and that of PR increased. We also observed a loss or significant decrease of the cytoplasmic and membrane-associated components of PR immunostaining. We suggest that using different tissue pretreatment protocols, we can distinguish between the 2 different forms of the 2 steroid hormone receptors. (The J. Histotechnol17:321, 1994)