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.
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.
Authors: S Arii; M Mise; T Harada; M Furutani; S Ishigami; M Niwano; M Mizumoto; M Fukumoto; M Imamura Journal: Hepatology Date: 1996-08 Impact factor: 17.425
Authors: Carlo Schneider; Andreas Teufel; Tetyana Yevsa; Frank Staib; Anja Hohmeyer; Gudrun Walenda; Henning W Zimmermann; Mihael Vucur; Sebastian Huss; Nikolaus Gassler; Hermann E Wasmuth; Sergio A Lira; Lars Zender; Tom Luedde; Christian Trautwein; Frank Tacke Journal: Gut Date: 2012-01-20 Impact factor: 23.059