Roderfeld M, Rath T, Lammert F, Dierkes C, Graf J, Roeb E. Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC. World J Hepatol 2010; 2(5): 175-179 [PMID: 21160992 DOI: 10.4254/wjh.v2.i5.175]
Corresponding Author of This Article
Elke Roeb, Professor, Justus-Liebig-University, Department of Medicine II, Gastroenterology, Paul-Meimberg-Str. 5, Giessen 35385, Germany. elke.roeb@innere.med.uni-giessen.de
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World J Hepatol. May 27, 2010; 2(5): 175-179 Published online May 27, 2010. doi: 10.4254/wjh.v2.i5.175
Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC
Martin Roderfeld, Timo Rath, Frank Lammert, Christian Dierkes, Jürgen Graf, Elke Roeb
Martin Roderfeld, Timo Rath, Elke Roeb, Justus-Liebig-University Giessen, Department of Medicine II, Gastroenterology, Giessen 35385, Germany
Frank Lammert, Department of Internal Medicine II, Saarland University Hospital, Saarland University, Homburg 66421, Germany.
Christian Dierkes, Justus-Liebig-University Giessen, Department of Pathology, Giessen 35385, Germany.
Jürgen Graf, Philipps-University Marburg, Department of Anaesthesiology and Intensive Care, 35033 Marburg and Passenger Medical Care, Deutsche Lufthansa AG, Frankfurt 60546, Germany
Author contributions: Roderfeld M designed the study, analyzed and interpreted data, and drafted the manuscript; Rath T and Dierkes C were responsible for acquisition of data, material, technical and intellectual support, analysis, and interpretation; Lammert F and Graf J provided critical revision of the manuscript and important intellectual content; Roeb E was responsible for study design, supervision and drafting of the manuscript.
Supported by the Grants from the Deutsche Forschungsgemeinschaft (RO 957/8-1 and SFB/TRR 57) and by BMBF ZooMAP-TPC4; a Research Grant of the University Medical Center Giessen and Marburg (UKGM 10/2010 GI)
Correspondence to: Elke Roeb, Professor, Justus-Liebig-University, Department of Medicine II, Gastroenterology, Paul-Meimberg-Str. 5, Giessen 35385, Germany. elke.roeb@innere.med.uni-giessen.de
Telephone: +49-641-9942338 Fax: +49-641-9942339
Received: January 8, 2010 Revised: April 9, 2010 Accepted: April 16, 2010 Published online: May 27, 2010
Abstract
AIM: To investigate the proteolytic contribution of tumor-associated macrophages (TAM) in tumor invasion, we analyzed whether TAM at the invasive front of small HCC in Abcb4-/--mice show an enhanced expression of MMP-9.
METHODS: Liver cryosections of the hepatocellular carcinoma (HCC) invasive front from 12 mo old Abcb4-/--mice were stained for collagen type I and MMP-9 using Alexa488 and Alexa568 labeled secondary antibodies. Afterwards, the Alexa568 dye was bleached and the macrophage marker F4/80 was visualized using Alexa568 labeled secondary antibodies. Finally, photographs of the invasive tumor front were digitally overlaid and analyzed.
RESULTS: After complete bleaching of the primary dye, specific fluorescence staining of a third antigen, here F4/80, was successfully performed on the same histological section. With this method, we were able to identify conglomerates of matrix metalloproteinase (MMP-9) expressing macrophages within the tumor capsule of HCC.
CONCLUSION: MMP-9 expressing macrophages are involved in matrix remodelling at the invasive tumor front of HCC. The described staining protocol provides a simple yet powerful extension of conventional immuno-histochemistry, facilitating visualization of at least three different antigens plus nuclei in one single histological section.
