Cyp8b1

Tissue cryosections (8 µm) were washed with PBS and fixed in 100% methanol for 10 min at room temperature. After three washes with PBS, the tissue sections were blocked in blocking buffer (3% BSA, 0.3% Triton™ X-100 in PBS) for 30 min at room temperature and then incubated with primary antibody overnight at 4 °C. The next day, the tissue sections were washed with PBS and incubated with fluorescence-conjugated secondary antibodies for 1 h at room temperature. DAPI-containing mounting medium (Southern Biotech #0100–20) was used to visualize the nuclei and preserve slides. The antibodies used were as follows: CYP8B1 (Affinity, DF4762; 1:200 dilution), Glutamine synthetase (BD Biosciences, #610517; 1:200 dilution), Cy3-conjugated Goat anti-rabbit IgG (Servicebio, GB21303; 1:200 dilution), and 488-conjugated Goat anti-mouse IgG (Servicebio, GB25301; 1:200 dilution). Images were acquired using a Nikon Eclipse C1 microscope and analysed using ImageJ software.