Envision hrp technique

IHC was performed by a two-step EnVision/HRP technique (Dako Cytomation, Denmark) according to the manufacturer's instructions. The following antibodies (Abcam, Cambridge, UK) were used for IHC: anti-human IL-33 monoclonal antibody diluted to 1:200; anti-human mast cell monoclonal tryptase diluted to 1:400; anti-human TGF-β1 polyclonal antibody diluted to 1:200; and anti-human IL-13 polyclonal antibody diluted to 1:200. All dilutions were made in 3% bovine serum albumin (BSA). Blank negative controls were conducted using 3% BSA instead of antibodies. Cell nuclei were counterstained using hematoxylin. For staining assessment, cells with brown-stained cytoplasm were defined as positive. A digital camera (Leica DM microscope) was used to take images of three randomly selected microscopic fields (leftup, middle, and rightdown, 20 × 10) for each slide. Quantitation of IL-33, TGF-β1, and IL-13 tissue expression was assessed by the size of positive staining area and relative to the control group, using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). The expression of MC in BA liver tissue was assessed by MC count in the portal area.

Immunohistochemistry method was performed using a two-step EnVision/HRP technique (Dako Cytomation, Denmark) according to the manufacturer's instruction as the description of protocol referenced to the publication [9 (link)]. The proteins included 3β-hydroxy steroid dehydrogenase (3β-HSD), CYP17, Cytochrome P450 aromatase (CYP19), AR, insulin-like growth factors (IGF-1), and growth differentiation factor-9 (GDF-9). Table 2 lists the antibodies used in this research. Two investigators assessed degree of immunostaining by blinded examination (Table 2).