Goat anti rabbit antibodies

For immunocytochemical staining, the following antibodies were used: rabbit anti-OR2H2 (ABIN6100219, Antikörper.de, Aachen, Germany) and rabbit anti-OR2W3 (PA5-61043, ThermoFisher Scientific). The antibody specificity has already been demonstrated using recombinant-expressed rho-tagged ORs in Hanna3A cells (Flegel et al., 2015 (link)). The thyroid cells were seeded on coverslips and maintained as previously described (Jovancevic et al., 2017a (link)). The cells were fixed through 20-min incubation at 4°C in 4% paraformaldehyde. Afterward, the cells were washed and permeabilized with a solution containing PBS −/− and Triton X-100 (PBST). The cells were blocked using PBST + 1% gelatin from cold-water fish skin (Sigma-Aldrich) and 10% goat serum. The primary antibodies (1:100 diluted) were incubated overnight at 4°C in PBST + 1% gelatin and 2% goat serum. To allow detection, cells were incubated with fluorescent goat anti-rabbit antibodies (1:1,000, ThermoFisher Scientific), Alexa Fluor™ 488 Phalloidin (1:200, ThermoFisher Scientific) and Hoechst 33258 (1:1,000, ThermoFisher Scientific), for 45 min in PBST + 2% gelatin and 2% goat serum. Cells were rewashed with PBST and mounted on a slide with ProLong™ Gold Antifade Mountant (ThermoFisher Scientific). Images were captured by using an LSM510 Meta confocal microscope (Zeiss, Jena, Germany).

Both non-transfected and transfected MCs were fixed with 4% paraformaldehyde for 30 min at room temperature and then permeabilized in PBS containing 0.1% Triton X-100 on ice for 10 min. And then samples were confined by 3% goat serum (Beyotime, Nantong, China) for 1 h at room temperature and incubated by overnight at 4 °C using anti-p50 antibody (Abcam, 1:100), anti-p65 antibody (Abcam, 1:100), anti-NLRP3 inflammasome antibody (Sangon Bio Tech, 1:50), anti-mcp-1 antibody (Sangon Bio Tech, 1:50), anti-TNF-α antibody (Bioworld Tech, Minnesote, CA, USA, 1:50), anti-IL-1β antibody (Bioworld Tech, 1:50), anti-Fn antibody (Sangon Bio Tech, 1:50) or anti-Col4 antibody (Proteintech, Wuhan, China, 1:50). Then, samples were incubated with dylight488 or dylight549 for fluorescent labeling of goat anti-rabbit antibodies (Thermo Fisher Scientific) for 1 h at 37 °C. Subsequently, cells were stained with DAPI for 10 min at room temperature. Finally, the images were observed with confocal microscope and analyzed with LAS AF Lite (Leica).

Cells grown on coverslips were washed gently with phosphate buffered saline (PBS) and fixed in 4% PFA (except that cells were fixed in cold methanol for endogenous ARHGAP31 staining), followed by permeabilization with 0.3% Triton X-100 in PBS. The cells were then blocked for 1 h in 5% goat serum, followed by incubation overnight with primary antibodies at 4°C. Anti-FLAG (Beyotime biotechnology, AF519, 1:500 dilution), anti-GM130 (Sigma-Aldrich, #610822, 1:200 dilution), anti-ARHGAP31 (CdGAP (G-8), Santa Cruz, sc-393839, 1:25 dilution), anti-HA (Beyotime biotechnology, AF0039, 1:200 dilution), and antibodies were used. Secondary antibodies were Alexa Fluor (488, 555)-labeled goat antimouse and goat antirabbit antibodies (Thermo Fisher, 1:500 dilution). DAPI was used to visualize the nuclei. Fluorescence images were captured with a 20× or 63× objective using a Zeiss LSM780 laser scanning confocal microscope.