The Use of Alkaline EDTA Solution Improves Heat-induced Epitope Retrieval for Immunohistochemical Localization of MIB-1 Antigen

This study was carried out to show whether the removal of tissue-bound calcium ions during a heat-induced epitope retrieval (HIER) procedure improved immunohistochemical staining of the tissues. In addition, the proteolytic susceptibilities of the fixed tissues before and after various epitope retrieval procedures were also evaluated. Immuno-histochemical staining for MIB-1 antigen from paraffin-embedded cases of tonsillitis, gastric adenocarcinomas, and breast carcinomas was performed following heat-treatments for 1, 2, 3, and 5 min in the presence of various buffers such as 0.01 M citrate buffer (pH 6.0), 0.1 M Tris-HCI buffer (pH 8.0), and 1 mM EDTA-NaOH solution (pH 8.0), each of which has different calcium chelating capabilities. In addition, the immuno-histochemical staining was performed following 0.05% and 0.1% trypsinizations of the tissues before and after the epitope retrieval. The trypsin-treated tissues were also analyzed with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunohistochemical staining of MIB-1 was most prominent when 1 mM EDTA solution (pH 8.0) was employed during HIER. Although trypsin digestion of the tissues before and after HIER generally increased the staining intensity, the cellular morphology of the background was poorly preserved. In addition, no significant differences were observed with gel electrophoresis of the trypsin digested tissues prepared in the various epitope retrieval solutions. Taken together, it can be suggested that the EDTA solution is more effective than other buffers in exposing epitopes presumably by removing tissue-bound calcium ions. Also, an additional trypsin treatment is not necessary even before and after the HIER as long as the EDTA solution is used.