Tgf β

For the detection of cytokines, inflammatory mediators and cell populations by immunohistochemistry, sections were treated as described before [21 (link)]. The slides were incubated overnight at 4°C with the primary antibodies that recognized HMGB1 (Abcam, UK; dilution 1:200), Caspase-3 (Abcam, UK; dilution 1:100), TGF-β (Abbiotec, CA, USA; dilution 1:300), MMP-9 (Santa Cruz Biotechnology, CA, USA; dilution 1:200), TNF-α (Santa Cruz Biotechnology, CA, USA; dilution 1:200) or IL-10 (Abbiotec, CA, USA; dilution 1:100) and prepared for visualization under a Nikon ECLIPSE E600 microscope.

For immunohistochemical studies, the paraffin-embedded tissues were cut (sections of 4 μm), deparaffinized in xylene and rehydrated with alcohol. Antigen retrieval was performed by heating the tissue in the presence of citrate buffer [15 (link)]. Tissues were blocked for endogenous peroxidase with 3% hydrogen peroxidase in methanol and rinsed in Tris-HCl (pH 7.4). To reduce non-specific binding, sections were incubated for 30 min at room temperature. Samples were then incubated over-night at 4°C with anti-human antibodies that recognize CD4 (Spring Bioscence, CA, USA), CD8 (DAKOCytomation, CA, USA), CD20 (Biocare Medical, CA, USA), CD68 (Biocare Medical, CA, USA), IFNγ (Abbiotec, CA, USA), TNFα (Abbiotec, CA, USA), IL-10 (Abbiotec, CA, USA), RANTES/CCL5 (Santa Cruz Biotechnology, CA, USA) or TGF-β (Abbiotec, CA, USA). The next day, sections were incubated with rabbit anti-mouse IgG, a secondary antibody horseradish peroxidase (HRP) conjugate (Spring Bioscience, CA, USA), for 30 min at room temperature. For negative control of the immunohistochemical reactions, samples were incubated only with the secondary HRP-conjugated antibody. Reactions were revealed with diaminobenzidine (Dako, CA, USA) as a chromogen and the sections were counterstained in Meyer’s hematoxylin (Dako). Finally, samples were examined under a Nikon ECLIPSE E600 microscope.