Bs 12867r

The frozen sections of the liver and ileum was fixed with 10% paraformaldehyde for 15 min. The sections were permeableised in 0.25% Triton X-100 and then blocked with 5% goat serum for 30 min. After incubation with primary antibody overnight at 4 °C and secondary antibody for 1 h at RT, the slides were washed thrice with PBS and then counterstained using DAPI. The following primary antibodies were used in this study: anti-NR1H4 antibody (Bioss, bs-12867R; 1:200); anti-CYP7A1 antibody (Bioss, bs-21430R, 1:200); and anti-CYP27A1 antibody (Abcam, ab126785, 1:200). The images were obtained by fluorescence microscopy.

The total protein isolation, denaturing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferring, blocking, incubation, visualization, and quantification assay were based on our published methods (21 (link)). The following antibodies were used: antibodies against FXR (1:500, bs-12867R, Bioss), TGR5 (1:500, NBP2-23669SS, Novus), SREBP1 (1:800, ab28481, Abcam), PPARA (1:1000, 66836-1-Ig, Proteintech), phosphor-PPARA (P-PPARA, S12) (1:800, ab3484, Abcam), and GAPDH (1:1,000, 2118S, Cell Signaling Technology).

According to the method of Xu et al. [31 (link)], 30 mg of hepatopancreatic tissue was dissected, PBS, cell lysate, protease inhibitor, and phosphorylase inhibitor were added, and finally, PMSF was added for disruption. The concentration of hepatopancreatic protein was measured using the BCA method and subsequently adjusted to a concentration of 4 mg/mL with PBS and loading buffer. Afterwards, the protein samples underwent denaturation in boiling water for a duration of 10 min before being utilized for the separation of proteins (36 mg of total protein per gel hole) through SDS-PAGE. After electrophoresis, they were transferred to membrane and block with rapid blocking solution for 25 min at room temperature. After development with ECL reagents, bands were quantified using Image J (version 1.43, National Institutes of Health). In this study, the following antibodies were used: antibodies against SREBP1(1:800, ab28481, Abcam), FXR (1:1500, bs-12867R, Bioss), GAPDH (1:1000, 2118 S, Cell Signaling Technology).