Anti txnip antibody

Cells grown on coverslips in a 6-well plate were fixed in 4% paraformaldehyde for 10 min after being rinsed with 1X PBS. Cells were then permeabilized with 0.2% Triton X-100 solution for 5 min. Then, cells were then blocked by 3% BSA (A3294, Sigma-Aldrich) for 1 hour, followed by overnight incubation with anti-CHOP antibody (1:400, #2895S, Cell Signaling Technology) and anti-TXNIP antibody (1:400, #14715S, Cell Signaling Technology) at 4°C. After that, cells were washed three times with 1× PBS and then incubated with the fluorescently labeled secondary antibody (1:400) for 1 hour in the dark at 37°C. Cells were then washed three times (protected from light) and counterstained with nuclear dye DAPI (Cat.D9542, Sigma-Aldrich). The slides were mounted by ProLong^TM Diamond Antifade Mountant (Cat.P36965, Invitrogen) and images captured by a Leica Microsystems confocal microscope.

Homogenized liver tissue specimens were boiled with 2 × SDS-PAGE loading buffer at 95 °C for 10 min. Then, proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred onto a PVDF membrane. After blocking with 5% nonfat dried milk in TBS-T (10 mM Tris-HCl, pH7.8, 150 mM NaCl and 0.05% [v/v] Tween-20) for 2 h at room temperature, the membrane was incubated with anti-TXNIP antibody (Cell Signaling Technology) or anti-β-actin antibody, diluted 2,000 to 5,000 fold, for at least 3 h at room temperature. The membrane was then washed and incubated with anti-rabbit secondary antibodies (5,000 to 10,000 dilution) for 1 h at room temperature. Protein bands were detected using the ChemiDoc XRS⁺ detection system (ECL, Bio-Rad); densitometric analysis was performed using Quantity One^® Image Analyzer (Bio-Rad).

Immunofluorescence and immunohistochemistry staining were performed on paraffin‐embedded or OCT‐embedded tissues fixed by 4% paraformaldehyde using standard techniques. The primary antibodies were used as followed: anti‐TXNIP antibody (1:200, 14715, Cell Signaling Technology), anti‐NLRP3 (15101, Cell Signaling Technology), anti‐caspase‐1 (24232/3866, Cell Signaling Technology), anti‐ASC (sc‐514414, Santa Cruz), anti‐GSDMD (NBP2‐33422, Novus Biologicals/ab219800, Abcam). The cell nuclei were incubated with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Sigma‐Aldrich). The images were examined using fluorescence microscopy or light microscopy (Olympus, Japan).