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Methods of Antigen Recovery Vary in Their Usefulness in Unmasking Specific Antigens in Immunohistochemistry

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°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/MIB1, 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 Ki-67/MIB1 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-1 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/MIB1. 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.