Ab234982

Liver tissues were fixed with 4% paraformaldehyde solution, embedded in paraffin blocks and processed by immunohistochemistry staining. Tissue sections were deparaffinized and rehydrated using a graded ethanol series and distilled water, and then treated with 3% H₂O₂ in methanol for 30 min to block endogenous peroxidase activity. Tissue sections were then rinsed twice for five minutes in phosphate-buffered saline (PBS) and incubated with 10% normal goat serum for 30 min to block non-specific antibody binding. After washing, the samples were incubated with primary antibodies against CYP7A1 (Abcam, ab234982, 1:500), CYP8B1 (Abcam, ab175843, 1:50), CYP27A1 (Abcam, ab126785, 1:250), and CYP7B1 (Abcam, ab175889, 1:100). Sections were then washed in PBS three times and incubated with secondary antibodies. The sections were stained with DAB according to the manufacturer’s protocol, mounted on slides, and photographed using a digital microscope camera (Nikon, Tokyo, Japan). The immunohistochemistry sample images were quantified using Image-Pro Plus software (Media Cybernetics, MD, USA).

HB (collection in Sichuan, 20042901), ZZ (collection in Fujian, 210303), DH (collection in Gansu, 20201211), and MX (collection in Jiangsu, 21032501) were obtained from Simcare Ltd. Primary antibodies against ZO-1 (21773-1-AP, 1:1000), occludin (13409-1-AP, 1:1000), claudin-1 (13050-1-AP, 1:1000), and the second antibody of beta-actin (20536-1-AP, 1:10000) were purchased from Proteintech Group Inc. Primary antibodies against NTCP (ab131084, 1:1000), BSEP (ab155421, 1:1000), CYP7A1 (ab234982, 1:1000), MRP2 (ab172630, 1:1000), and FXR1 (ab129089, 1:1000) were obtained from Abcam Inc. ANIT (N106389), UDCA(U110695), and olive oil (O108685) were purchased from the Aladdin Chemical Reagent Co. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (reference D4540) (St-Louis, MO, USA). The TRIzol total RNA extraction kit was obtained from Life Technologies.

Ileum and liver tissues were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, Tullagreen, Ireland). The membranes were blocked, and then incubated with primary antibodies against FGF15 (1:800, sc-398338; Santa Cruz Biotechnology, USA), SHP (1:800, A1836; ABclonal Technology, Wuhan, China), CYP7A1 (1:1000, ab234982; Abcam, Cambridge, UK), CYP7B1 (1:1000, 24889-1-AP; Proteintech, Rosemont, IL, USA), SREBP2 (1:1000, ab30682; Abcam, Cambridge, UK), ABCG5 (1:1000, 27722-1-AP; Proteintech, Rosemont, IL, USA), ABCG8 (1: 1000, DF6673; Affinity Biosciences, Changzhou, China), and GAPDH (1:5000, 60004-1-Ig; Proteintech, Rosemont, IL, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase conjugated secondary antibodies. The blots were developed using the enhanced chemiluminescence (ECL) method. The gray values of the bands were calculated using ImageJ software and were normalized to GAPDH. The relative expression levels were shown as fold changes relative to control group.