Cy3 goat anti rabbit secondary antibody

Each sciatic nerve sample was homogenized and total protein was extracted. Protein concentrations were measured. Ten samples of each group underwent electrophoresis, and then transferred onto the polyvinylidene fluoride membrane. Membranes were blocked with 5% skimmed milk, incubated with rabbit anti-rat type I collagen monoclonal antibody (1:200; Abcam, Cambridge, UK) and rabbit anti-rat type III collagen monoclonal antibody (1:400; Abcam) in a wet box overnight at 4°C, followed by 0.5 hours at room temperature. Then they were incubated with CY3 goat anti-rabbit secondary antibody (1:200; Abcam) in the dark for 1 hour at room temperature, and visualized with X-ray. Optical densities of types I and III collagen were analyzed with Image-Pro Plus 5.0.2.9 software (Media Cybernetics, Bethesda, MD, USA). Results were presented by the ratio of optical density of collagen to β-actin.

Briefly, brain sections were incubated in citrate buffer at 60°C for 30 minutes and in 5% goat serum at room temperature for 1 hour. The sections were then incubated with rabbit anti-CD206 primary antibody (a marker for M2 microglia; 1:500, Abcam, Cat# ab64693, RRID: AB_1523910) at 4°C overnight and Cy3 goat anti-rabbit secondary antibody (1:500, Abcam, Cat# ab6939, RRID: AB_955021) at room temperature for 1 hour. Finally, anti-fluorescence quenching sealing tablets containing 4,6-diamidino-2-phenylindole were used to stain the nuclei, and fluorescent images were taken using a fluorescence microscope (Nikon, Tokyo, Japan).
BV2 cells were treated with the drugs for 24 hours, and then were fixed with 4% paraformaldehyde for 20 minutes, followed by permeabilization and blocking with 0.1% Triton X-100 and 5% donkey serum (Solarbio) at room temperature for 1 hour. The cells were then incubated with rabbit anti-NLRP3 antibody (1:500, Abcam, Cat# ab214185, RRID: AB_2819003) at 4°C overnight followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:500, Abcam, Cat# ab150073, RRID: AB_2636877) at room temperature for 2 hours. Finally, 4,6-diamidino-2-phenylindole was used to stain the nuclei, and fluorescent images were taken. The level of NLRP3 was observed using a fluorescence microscope.