Sc 32294

Oesophageal strips or cultured cells were collected to extract the protein with RIPA lysis buffer (Beyotime Biotech), which contained 1 mmol/L final concentration of phenylmethanesulfonyl fluoride (PMSF). After complete lysis, the samples were centrifuged at 10 000 g for 5 minutes at 4°C to precipitate the tissue debris. The supernatants were used to measure the protein concentration by a BCA Protein Assay Kit (Beyotime Biotech). The proteins were electrophoresed in 10% SDS‐PAGE gels and then transferred to PVDF membranes. After blocking with 5% skim milk for 1 hour at room temperature, the membranes were incubated with the following primary antibodies: CaSR (1:200, ab19347, Abcam), NLRP3 (1:100, ab214185, Abcam), ASC (1:100, sc‐22514‐R, Santa Cruz), Caspase‐1 p20 (1:100, sc‐398715, Santa Cruz) and IL‐1β (1:100, sc‐32294, Santa Cruz) at 4°C overnight. The membrane was washed with TBST and incubated with secondary antibodies for 2 hours at room temperature. Protein bands were visualized on the membrane with a Gel Imaging System, and the protein bands were quantified with ImageJ software.

ESCs and CSCs were cultured on coverslips, and the medium was replaced with SFM containing MCC950 (100 μM). After incubation for 16 h, the cells were fixed in methanol for 2 min at room temperature and permeabilized with 0.5% Triton X-100 for 1 min. After blocking with 1% bovine serum albumin for 1 h at room temperature, the cells were incubated with primary antibodies against NLRP3 [19771–1-AP, 1:200, Proteintech, Chicago, IL, USA], IL-1β [sc-32294, 1:100, Santa Cruz Biotechnology, Heidelberg, Germany], CD10 [sc-9149, 1:50, Santa Cruz Biotechnology], vimentin [sc-6260, 1:50, Santa Cruz Biotechnology], and fibronectin [ab2413, 1:200, Abcam, Cambridge, UK] for 2 h at room temperature. The cells were then incubated with goat anti-rabbit (Alexa Fluor® 568, 1:500, Thermo Fisher Scientific, for anti-NLRP3 antibody) and goat anti-mouse (Alexa Fluor® 488, 1:500, Thermo Fisher Scientific, for anti-IL-1β antibody) secondary antibodies for 1 h at room temperature. Nuclear staining was carried out using 4',6-diamidino-2-phenylindole (4083, 1:1000, Cell Signaling Technology). Visualization was performed using a confocal laser-scanning microscope (BZ9000, Keyence, Osaka, Japan).

Immunohistochemical localization was made, as previously described by Esposito et al. [40 (link)]. Slices were incubated at room temperature overnight with one of the following primary antibodies: Anti-ZO1 (617300 Invitrogen, 1:200 in PBS, v/v), anti-occludin (71-1500 Invitrogen, 1:200 in PBS, v/v), anti-TNF-α (sc-52746 Santa Cruz Biotechnology, 1:100 in PBS, v/v) anti-IL-1β (sc-32294 Santa Cruz Biotechnology, 1:50 in PBS, v/v) and anti-iNOS (610432 BD Transduction, 1:50 in PBS, v/v). At the end of the incubation with the primary antibody, the sections were washed with PBS and incubated with a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The reaction was revealed by a chromogenic substrate (brown DAB), and counterstaining with NUCLEAR FAST-RED. A negative control was performed using no primary antibody, particularly, tissue was incubated with the antibody diluent alone, followed by incubation with secondary antibodies and detection reagents.
All stained sections were observed and analyzed as previously described. For immunohistochemistry 20× (50 µm scale bar) and 40× (20 µm scale bar) were shown.