Microwave heating of histologic sections in
citrate buffer (MAR) is a widely used method of
antigen recovery but often results in loss of tissue sections. Low-temperature
antigen retrieval (LTAR), incubation at 80 degrees C in
citrate buffer for 2 hours with
trypsin pretreatment is an alternative method reported to result in better
antigen recovery for specific
antigens as well as decreased loss of tissue sections. To optimize our immunohistochemical evaluation of
breast carcinomas, we compared the efficacy of these methods of
antigen recovery for several important
antigens. Ten
breast carcinomas were immunostained for
estrogen and
progesterone receptors (ER and PR), Ki-67/ MIB 1, p27/Kip-1, and Bcl-2 after MAR, LTAR with enzymatic pretreatment, or no
antigen recovery. The immunohistochemical staining was scored and compared for each antibody and
antigen recovery combination. The proportion of tissue lost from each slide after staining also was assessed. More and stronger positive staining was achieved with
antibodies to Ki67/MIB 1 and ER when LTAR was used compared with the other two methods; in contrast, optimal staining with
antibodies to Bcl-2 was achieved when MAR was used. Staining with anti-p27/Kip- was nearly equal with either LTAR or MAR. Staining with anti-PR was slightly better with MAR than with LTAR. Tissue loss was greatest for MAR compared with LTAR or with no
antigen recovery. For selected cases, LTAR caused focal tissue damage, and either the immunostaining with LTAR had to be repeated or only a portion of some tissue sections would be used for examination. LTAR was the most effective for ER and Ki-67/MIB 1. MAR provided the most intense staining for Bcl-2 and PR, but this enhanced staining must be weighed against the greater tissue section loss from MAR. This study demonstrated that AR methods are not equally applicable to all
antibodies.