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

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概要

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• 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-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 immunohistochemical staining was performed following 0.05% and 0.1% trypsinzations of the tissues before and after the epitope retrieval. The trypsin-treated tissues were also analyzed with sodium dodecylsulfatepolycarylamide 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. Altough 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 tissueboud calcium ions. Also, an additional trypsin treatment is not necessary even before and after the HIER as long as the EDTA solution is used.

• 日本組織細胞化学会の論文

著者

• LEE Sang

  Department of Pulmonary and Critical Care Medicine, Gachon University, Gil Medical Center

• PAIK Seung

  Department of Pathology, Hanyang University School of Medicine

• Kim Young-Sik

  Department of Game and Multimedia Engineering, Korea Polytechnic University

• Lee Sang

  Department Of Advanced Materials Science And Engineering Mokpo National University

• Lee Sang

  Department Of Pathology College Of Medicine Korea University Ansan Hospital

• Kim Y‐s

  Korea Univ. Seoul Kor

• KIM Insun

  Departments of Pathology, Orthopedic Surgery and Radiology, College of Medicine, Korea University

• Kim Insun

  Department Of Biology College Of Natural Sciences Kyungpook National University

• Kim In-mook

  Fakultat 4 Theoretische Physik Gerhard-mercator-universitat Duisburg

• Kim Insun

  Department Of Pathology College Of Medicine

• Paik Seung

  Department Of Biochemistry College Of Medicine Inha University

• Kim Young-sik

  Gastrointestinal Research Laboratory Veterans Administration Medical Center University Of California

• Kim Gyoung-Mi

  Department of Biochemistry, College of Medicine, Inha University

• Kim Gyoung-mi

  Department Of Biochemistry College Of Medicine Inha University

• Kim I

  Lg Cable Co. Ltd. Kyunggi‐do Kor

• Kim Young-sik

  Department Of Pathology College Of Medicine

• Kim Young-sik

  Department Of Computer And Radio Communications Engineering Korea University

• LEE Sang

  Department of Acupuncture & Moxibustion, College of Oriental Medicine, Kyung Hee University

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