title: Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Martin Roderfeld, Timo Rath, Elke Roeb, Justus-Liebig-University Giessen, Department of Medicine II, Gastroenterology, Giessen 35385, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Roderfeld
authors.forename: Timo
authors.initials: T
authors.lastname: Rath
authors.forename: Frank
authors.initials: F
authors.lastname: Lammert
authors.forename: Christian
authors.initials: C
authors.lastname: Dierkes
authors.forename: Jürgen
authors.initials: J
authors.lastname: Graf
authors.forename: Elke
authors.initials: E
authors.lastname: Roeb