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 typeⅠand 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 Alexa 568 dye was bleached and the macrophage marker F4 / 80 was 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. 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 macrophag es are involved in matrix remodeling 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.