Anti fxr

Formalin-fixed paraffin kidney sections were deparaffinised and incubated with the polyclonal primary antibody anti-FXR (1:50 dilution, Abcam Ltd, Cambridge), anti-fibronectin (dilution 1:1,000, Abcam) and anti-collagen IV (dilution 1:1,000, Abcam) overnight, followed by horseradish peroxidase anti-rabbit Envision system (Dako Cytochemistry, Tokyo, Japan) the following day. Staining was developed with 3.3diaminobenzidine tetrahydrochloride (Dako Cytochemistry, Tokyo, Japan) before counterstaining with Mayer’s haematoxylin stains. Antibody against rabbit IgG was used as a negative control. Images were analysed using an Olympus microscope (Olympus, Japan) and Image J software.

Tissue slides were incubated at 60 °C for 4 h before deparaffinization and rehydration. The slides were boiled for 2 min in a pressure cooker filled with sodium citrate to retrieve antigen. Anti-FXR (#187735, 1:50, Abcam, UK) and anti-SNAI2 (#PA5–73015, 1:50, Invitrogen, USA) antibodies were dripped onto the slides and incubated overnight at 4 °C. Subsequently, slides were incubated with secondary antibodies for 40 min at room temperature. Finally, the sections were stained with DAB and hematoxylin, dehydrated and transparentized. The results obtained were scored by two professional pathologists independently. The scoring was the product of the positive rate and intensity of staining. The staining intensity was scored as follows: negative (0), weak (1), moderate (2), and strong (3). The positive rate of staining was scored as <10% (0), 10–25% (1), 26–50% (2), 51–75% (3), and >75% (4).

Total protein was extracted by mixing cells with radioimmunoprecipitation assay (RIPA) buffer (#P0013B, Beyotime, China), which was supplemented with protease and phosphatase inhibitors. Next, the proteins were separated by SDS-PAGE and transferred to NC membranes (#66485, Pall Inc., USA). The resulting membranes were incubated with 5% milk/TBST for 2 h at room temperature. Next, TBST was used to wash the membranes for 3 × 5 min, after which they were incubated with primary antibodies at 4 °C overnight. Finally, the membranes were incubated with HRP-conjugated secondary antibodies (#SAG10002, AntiProtech Inc., USA) after washing in TBST for 3 × 7 min. The following antibodies were used: anti-FXR (#187735, 1:200, Abcam, UK), anti-SNAI2 (#106077, 1:1000, Abcam, UK), anti-HDAC6 (#7558, 1:1000, Cell Signaling Technology, USA), anti-HNF4α (#92378, 1:2000, Abcam, UK), anti-CDX2 (#12306, 1:1000, Cell Signaling Technology, USA), and anti-β-actin (#AP0060, 1:5000, Bioworld, China). The primary antibody dilution buffer, PREstain Protein Ladder and Western SuperSensitive Substrate were purchased from BioCytoSci (#IC-8008, #IC-8001, China).