Use of pH 9.5 Tris-HCI Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry

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Abstract
Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies “routinely” used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCI buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCI buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HC1 containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HC1 with urea was also superior to Tris-HC1 alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HC1 with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HC1 with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.