The use of alkaline EDTA solution improves heat-induced epitope retrieval for immunohistochemical localization of MIB-1 antigen

Young Sik Kim, Sang Ju Lee, Insun Kim, Gyoung Mi Kim, Seung R. Paik

    Research output: Contribution to journalComment/debatepeer-review

    4 Citations (Scopus)


    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. Immunohistochemical 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-HCl buffer (pH 8.0), and 1 mM EDTA-NaOH solution (pH 8.0), each of which has different calcium chelating capabilities. In addition, the immunohistochemical staining was performed following 0.050% 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.

    Original languageEnglish
    Pages (from-to)287-294
    Number of pages8
    JournalActa Histochemica et Cytochemica
    Issue number3
    Publication statusPublished - 1999


    • Calcium
    • EDTA
    • Heat-induced epitope retrieval
    • Immunohistochemistry
    • Trypsin

    ASJC Scopus subject areas

    • Pathology and Forensic Medicine
    • Biochemistry
    • Physiology
    • Histology
    • Cell Biology


    Dive into the research topics of 'The use of alkaline EDTA solution improves heat-induced epitope retrieval for immunohistochemical localization of MIB-1 antigen'. Together they form a unique fingerprint.

    Cite this