Monoclonal rabbit anti txnip antibody

Firstly, the paraffin section was deparaffinised and then rehydrated. The paraffin section was further quenched in 3% hydrogen peroxide for 20 min and then incubated in goat serum for 30 min. The slides were incubated at 4°C overnight with polyclonal rabbit anti-BIP antibody (Abcam, USA), monoclonal rabbit anti-TXNIP antibody (Abcam, USA), or monoclonal rabbit anti-NLRP3 antibody (Cell Signaling Technology, USA).
The slides were rinsed with PBS and then incubated with goat anti-rabbit IgG for 30 min at 37°C. DAB (ZSGB-BIO; China) was used as the chromogen and hematoxylin (Sigma, USA) was used for nuclear counterstain. For negative controls, the primary antibodies were omitted. Experiments were repeated at least three times. Images were acquired using EVOS FL Auto Imaging System (Life Technologies, USA).

Treated HTR-8/SVneo cells were lysed with RIPA buffer (Beyotime, China). Protein concentration was determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, China), according to the manufacturer's instructions. Protein samples (20 μg) were loaded on 10% SDS-polyacrylamide gels, resolved by electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, USA). Immunoblotting was performed using polyclonal rabbit anti-BIP antibody (Abcam, USA), monoclonal rabbit anti-TXNIP antibody (Abcam, USA), monoclonal rabbit anti-NLRP3 antibody (Cell Signaling Technology, USA), polyclonal rabbit anti-ASC antibody (Santa Cruz, USA), or polyclonal rabbit anti-Caspase-1 antibody (Santa Cruz, USA) and β-actin (ZSGB-BIO, China). Following incubation with the secondary antibodies (Beyotime, China), the bands of specific proteins on the membranes were developed with Immobilon Western Chemiluminescent HRP Substrate (MILLIPORE, USA). The levels of proteins were quantified by a ChemiDoc™ XRS+ (Bio-Rad, USA). β-actin was used as the loading control.