Immunohistochemical localization of α-sma and TGF-β was performed as previously described [82 (link)]. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min [83 (link)]. The sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 20 min [84 (link),85 (link)]. Non-specific adsorption was minimized by incubating the section in 2% normal goat serum in phosphate-buffered saline for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were incubated overnight with primary antibodies: anti-α-sma antibody (Santa Cruz Biotechnology, sc-32251) or anti- TGF-β antibody (Santa Cruz Biotechnology, sc-130348). All sections were washed with PBS and then treated as previously reported [86 (link),87 (link)].
Samples were washed with PBS and incubated with secondary antibody (Vectastain Elite, PK-6200, Vector Laboratories, Burlingame, CA, USA). Specific labeling was identified with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). The stained sections were observed using a Leica (Wetzlar, Germany) DM6 microscope following a typical procedure [88 (link)].
Samples were washed with PBS and incubated with secondary antibody (Vectastain Elite, PK-6200, Vector Laboratories, Burlingame, CA, USA). Specific labeling was identified with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). The stained sections were observed using a Leica (Wetzlar, Germany) DM6 microscope following a typical procedure [88 (link)].