Prolong gold antifade reagent containing dapi

The immunofluorescence assay was performed with modifications, as previously described [45 (link)]. Briefly, cryosections and BMDM were washed with tris-buffered saline (TBS) and fixed in 4% paraformaldehyde (PFA) for 10 min. After washing, the samples were permeabilized with TBST (0.2% Triton X-100 in TBS) for 10 min and washed three times with TBS for 5 min. Samples were blocked using 2% BSA and incubated at 4°C overnight with primary antibodies, including rabbit anti-Nogo-A (Abcam, catalog no. ab62024), mouse anti-CD68 (Santa Cruz Biotechnology, catalog no. ab955), mouse anti-iNOS (Santa Cruz Biotechnology, ab49999), mouse anti-CD206 (Santa Cruz Biotechnology, catalog no. sc-58986), mouse anti-CHOP (Santa Cruz Biotechnology, sc-71136), and mouse anti-calnexin (Novus Biologicals, catalog no. NB300518). After three washes with TBS for 5 min, samples were incubated with secondary antibodies (donkey anti-mouse immunoglobulins (Alexa Fluor 488, Abcam, catalog no. ab150105) and donkey anti-rabbit immunoglobulins (Alexa Fluor 555, Abcam, catalog no. ab150066) for 1 h in the dark. The samples were mounted using ProLong™ Gold Antifade reagent containing DAPI to visualize the nuclei (Cell Signaling Technology, catalog no. 8961s) and were analyzed by confocal microscopy (ZEISS).

For β-catenin immunostaining, HCoEpiC cells were grown on an eight-chambered slide overnight. DCA/LCA (100 µM) were then treated with cells for 24 h and incubated for 72 h. Cells were fixed with paraformaldehyde for 15 min, washed three times with PBS, and then cells were permealized with ice-cold methanol for 5 min and washed three times with PBS. Cells were blocked with 5% normal goat serum in PBS at room temperature for 1 h and then incubated with anti-β-catenin antibody (1:100 dilution; Cell Signaling) overnight at 4 °C. After washing with 1 × PBS, cells were incubated with anti-rabbit IgG–FITC conjugate (1:100 dilution) antibody for 30 min at room temperature. Cells were washed with PBS and then placed on cover slips with prolong gold antifade reagent containing DAPI (Cell Signaling Technology, Boston, MA, USA). Cells were photographed utilizing an Olympus microscope and an Olympus microscope digital camera with DP2-BSW software.