Dimethylaminoazobenzene dab

The livers of the experimental groups were collected at 48, 72, 168, and 336 h post treatment. The specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5-μm sections. After hematoxylin–eosin (H&E) staining, liver pathological findings and images were assessed under a microscope (Leitz Aristoplan, Wetzlar, Germany). For immunohistochemical analysis, the liver sections at days 7 and 14 were stained with the anti-α-smooth muscle actin (α-SMA) antibody (1:200, Abcam, Cambridge, MA, USA), and then, the signals were visualized with the substrate dimethylaminoazobenzene (DAB) (Vector Laboratories, Burlingame, CA, USA). The α-SMA expression level was quantified in six to eight random microscopic fields using Image Pro Plus software.

Immunohistochemical staining was carried out using the VECTASTAIN ABC Kit (Vector Laboratories, Burlingame, CA, USA) in accordance with the manufacturer's instructions. Samples were treated with 0.5% H₂O₂ to block endogenous peroxidase activity for 10 min and permeabilized with 0.3% Triton‐X100. Non‐specific binding was inhibited by incubating samples with 1% normal horse serum for 1 h at room temperature (RT). Primary mouse anti‐CD31, anti‐F4/80 and anti‐SDF‐1α antibodies (Abcam, Cambridge, MA, USA) were added. After three PBS washes, samples were incubated with a biotin‐conjugated secondary antibody for 1 h at RT. After washing with PBS, the substrate solution was added and the reaction was allowed to proceed at RT for 40 min. To visualize the reactive area in the tissue, samples were treated with dimethyl‐aminoazobenzene (DAB; Vector Laboratories), counterstained with Fast Red (Vector Laboratories) for 10 min and mounted. Finally, samples were observed using a Nuance Multiplex Biomarker Imaging System (Cambridge Research Instrumentation, Woburn, MA, USA).