Anti α sma antibody clone 1a4

The engrafted tumors were fixed, stained with hematoxylin and eosin. The number of mitotic cells in microscopic 10 high power fields, ×400, (10 HPF) was counted. Immunohistochemical staining was performed according to the avidin-biotin complex (ABC) method. All staining processes from deparaffinization to counterstaining with hematoxylin were performed using the automated LEICA BOND-IIITM staining system (Leica Biosystems, Heidelberg, Germany). Antigen retrieval was not performed for α-SMA, but for vimentin, antigen retrieval was performed for 30 minutes by placing the sections in epitope retrieval buffer (pH 6) in the autostainer. The anti-α-SMA antibody (clone 1A4, code M0851, Dako, Glostrup, Denmark) was used at 1:150 dilution for a total reaction time of 15 minutes, while the anti-human multi-cytokeratin antibody (code NCL-L-AE1/AE3, Leica Biosystems) (1:300 dilution, 15 minutes) and the anti-human vimentin antibody (Clone V9; code M0725, Dako) (1:600 dilution, 15 minutes) were used to confirm the presence of human cell-derived tumors.

Liver tissues were fixed with 10% formalin, embedded in paraffin and cut into 4-μm sections stained with hematoxylin-eosin (H&E). Necroinflammatory activity in hepatic tissue was graded by a “blinded” liver pathologist according to the Scheuer scoring system [18 (link)]. For quantitative assessment of fibrosis, sections were stained with Sirius Red for quantitative analysis of collagen content [19 (link)]. α-SMA, a marker of hepatic stellate cells activation, was assessed by staining with rabbit anti-α-SMA antibody (clone 1A4; Dako, Glostrup, Denmark) and visualized with HRP labeled anti-rabbit antibody (ChemMate™ EnVision™ Detection Kit, Dako).

For myofibroblast immunodetection, deparaffinized histological slides 3, 6, and 9 were subjected to endogenous peroxidase activity blockade with 3% hydrogen peroxide and methyl alcohol (10 min in a dark room). Subsequent antigen recovery was performed by moist heat under pressure in 10-mM citrate buffer/pH 6.0 solution. The histological sections were incubated with anti-α-SMA antibody (clone 1A4, Dako, 1:100) for 30 min. The secondary antibody (SABC (streptavidin–biotin complex), catalog number SA1022) was incubated at 37 ℃ for 30 min. The reaction was revealed by incubating the histological slides with diaminobenzidine (DAB, Ventana Medical Systems, Tucson, AZ, USA) in a dark room for 30 min. Counterstaining was performed with Meyer’s hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). Two histological slides of myofibroma were used as a positive control, whereas the negative control was obtained using two other slides from the same tumor, replacing the primary antibody by TRIS–HCl. The number of α-SMA-positive cells was counted in ten field histological series of each histological Sect. (400 × , analytical area corresponding to 0.025 mm²). Final data were expressed as mean ± standard deviation (SD) cells/field.