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. 2010 May 27;2(5):175-9.
doi: 10.4254/wjh.v2.i5.175.

Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC

Martin Roderfeld  1 , Timo Rath, Frank Lammert, Christian Dierkes, Jürgen Graf, Elke Roeb
Affiliations

Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC

Martin Roderfeld et al. World J Hepatol. .

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.

Keywords: Fluorescence staining; Hepatocellular carcinoma; Matrix metalloproteinase; Tumor associated macrophages.

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Figures

Figure 1
Figure 1
Macroscopic and histological aspect of HCC in BALB/c-Abcb4-/--mice. A: Representative macroscopic view of BALB/c-Abcb4-/--mouse liver at one year of age. Typically, one prominent tumor and a number of smaller tumors are visible; B: Hematoxylin and Eosin (H&E) stained liver section of the invasive tumor front (tumor on the upper right, fibrotic parenchyma on the lower left side, dashed line: HCC tumor capsule, bar represents 100 μm); C: Fifty-fold enlarged Sirius red (SR) stained liver section photographed under polarized light (PL, bar represents 400 μm). Fibrillar collagens appear in red. The tumor capsule is rich in collagen, whereas tumor stroma appears collagen-free.
Figure 2
Figure 2
Conglomerates of tumor associated macrophages express MMP-9 within the HCC tumor capsule. A: Immunofluorescence co-staining of collagen type I (green) and MMP-9 (red) displayed clusters of MMP-9 expressing cells in the tumor capsule. B: After photobleaching of the red fluorescence dye, macrophage marker F4/80 was visualized. The long arrows indicate MMP-9 positive macrophages. The shot arrow indicates an MMP-9 expressing cell which is negative for F4/80. DAPI was used for nucleus staining. The left panels provide a section overview with 200-fold magnification and bars represent 100 μm (left side of the panel: Tumor; dashed line: Tumor capsule; box: This is enlarged to 1000 × magnification in the corresponding panel, bars represent 20 μm). Original green and red channel micrographs (1000 ×) were shown. Images were derived by conventional wide field fluorescence microscopy. Representative digital overlays are shown.
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