Anti rat nlrp3 antibody

Paraffin wax-embedded tissue sections of subchondral bone were used for IHC. The sections were dewaxed in xylene and dehydrated with gradient ethanol. After antigen retrieval in sodium citrate buffer, the sections were incubated with anti-rat NLRP3 antibody (Abcam, USA) overnight at 4 °C or at 37 °C. After three washes in PBS (3 min each time), the tissues were incubated with biotinylated IgG (1:200, Solarbio, China) at 37 °C for 30 min, followed by 3 times PBS rinse. Afterward, fresh diaminobenzidine (DAB) (Boster Biological Technology, China) was added for coloration for 1~2 min to visualize the antibody-antigen complexes. The slices with brown or yellow cytoplasm were considered to be positive. Five typical fields were randomly selected from each slice for observation and counted under an optical microscope (Nikon, Tokyo, Japan).

The chondrocytes, cultured on cover slips for 48 h, were fixed with 4 % paraformaldehyde solution (Santa Cruz Biotechnology, USA), followed by treatment with blocking solution containing 5 % donkey serum and 0.1 % Triton (Sigma Aldrich, Germany) in PBS. Subsequently, the samples were incubated with the polyclonal anti-Rat NLRP3 antibody (1:60, abcam, USA) overnight at 4 °C followed by washing three times with PBS and incubating with the anti-mouse IgG secondary antibody (1:200, abcam, USA) and DAPI (Roche Applied Science, Germany) for 1 h at room temperature. The images were collected by an Olympus FluoView FV10i confocal microscope.