Ab62698

Patient biopsies and porcine spine punches were fixed in 4% buffered formalin and paraffin embedded. Sections (2 μm) were stained with hematoxylin and eosin (H&E), Brown–Brenn (BB), Safranin-o, and Fast-green or Gram-staining. For immunohistochemistry (IHC), formalin-fixed paraffin-embedded tissue sections were pre-treated with the BOND Epitope Retrieval Solution 2 (Leica Biosystems) at 100°C for 30 min. They were stained with anti-cleaved caspase-3 antibody (polyclonal, ab2302, abcam) and caspase-1 antibody (polyclonal, ab62698, abcam) for 30 min. For detection, the slides were stained with the Bond Polymer Refine Detection HRP Kit (Leica Biosystems), according to the manufacturer’s instruction, and counterstained with hematoxylin. Whole-slide scanning and photomicrography were performed with a 108NanoZoomer 2.0-HT digital slide Scanner (Hamamatsu, Houston, TX, USA).

The total protein was extracted from placental tissues and HTR-8/SVneo cells using RIPA lysate (Beyotime, China) (with PMSF-protease and phosphatase inhibitor added), and then the protein concentration was measured by the BCA assay kit (Beyotime, China) according to the instructions of manufacturer. The protein samples of 30ug per well were added to 10% of the twelve-alkyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slot to separate the protein and then electrotransferred onto the PVDF membranes. Next, the nonspecific site of the membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4 ℃ with first antibodies NLRP3 (1:1000, ab214185, Abcam), Caspase-1 (1:500, ab62698, Abcam), GSDMD (1:500, sc-81,868, Santa Cruz) and β-actin (1:1000, AF0003, Beyotime). On the second day, the membranes were incubated with the horseradish peroxidase (HRP) labeled secondary antibodies (1:1000, A0208/A0216, Beyotime) at room temperature for 1 h. Finally, under the action of ECL luminescent liquid, immunoreactive signals were detected by the Bio-Rad gel imaging system (MG8600 Thmorgan Biotechnology Co., Ltd., Beijing, China). The IPP6.0 software (Media Cybernetics, Singapore) was used to analyze the gray values and calculate the relative protein levels of NLRP3, Caspase-1 and GSDMD. The experiment was repeated at least three times.

The liver tissues in each group were ground, and the hepatic cells in each group were harvested and washed with PBS. Total proteins were extracted using a protein extraction kit (BestBio; BB-3101), and the protein concentration in each extract was determined using the bicinchoninic acid (BCA) method. Next, a 20 μg aliquot of total protein from each extract was separated by 10% SDS-PAGE performed at 120 V. The separated protein bands were electrophoretically (200 mA for 90 mins) transferred onto PVDF membranes (Roche, Basal Switzerland, cat. no. 3010040001), which were subsequently blocked with 5% powdered skim milk. Next, the PVDF membranes were incubated with primary antibodies at 4°C overnight. After washing, the PVDF membranes were soaked with an HRP-labeled secondary antibody for 1 h and the immunostained protein bands were detected using the ECL chemiluminescence reagent (Millipore; KLS0500). The primary antibodies used in the study were as follows: NLRP3 (1 : 1000, Abcam, Cambridge, UK, ab214185), ASC (1 : 1000, Abcam, ab180799), and caspase 1 (1 : 1000, Abcam, ab62698).

After extracting the total proteins from cells and tissues, the separation was performed by 10% SDS-PAGE [22 (link)]. Then, the proteins were transferred to the PVDF membrane, followed by blocking for 1 h. Primary antibodies including anti-caspase-1 (ab62698, 1 : 1000, Abcam), anti-ASC protein (13833, 1 : 1000, Cell Signaling Technology), anti-GSDMD (39754, 1 : 1000, Cell Signaling Technology), anti-NLRP3 (13158, 1 : 1000, Cell Signaling Technology), and GAPDH (ab8245, 1 : 3000, Abcam) and the corresponding secondary antibodies were used. A hypersensitive ECL (BiossciBio, Hubei, China) was used for detection.