Af0693

Hippocampal tissues were ground and protein was extracted in RIPA buffer under low-temperature conditions, and the protein level was determined using the supernatant. Proteins (30 μg) were separated by electrophoresis on 10% SDS-PAGE gels using a flat gel apparatus, then transferred onto PVDF (0.45 mm) membranes and blocked with 5% skim milk for 1.5 h. The primary antibodies, mainly including NLRP3 (PAB38738, Bioswamp, 1:1000), MARK4 (AF0693, Affinity, 1:1000), PSD95 (#48638, signalwayantibody, 1:1000), SYN1 (A17362, ABclonal, 1:1000), SYP (#54990, signalwayantibody, 1:1000), Bcl-2 (bs-0032R, Bioss, 1:1000), GAPDH (GB15002, Servicebio, 1:1000)and Beta Actin (#52901, signalwayantibody, 1:10000)were incubated overnight at 4 °C, followed by incubation with appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. The protein bands were visualized using ECL chemiluminescent peroxidase substrate and quantified using the ChemiDoc XRS system (Bio-Rad, USA), and the immunoblot bands were quantitatively analyzed using Image J software.

For brain tissue paraffin sections, after 1 h of dewaxing, antigen retrieval was performed with sodium citrate solution at 90 °C for 15 min, followed by 45 min of blocking with 10% BSA for immunostaining at room temperature, overnight incubation with primary antibodies at 4 °C, and washing three times with PBS, then incubation with appropriate secondary antibodies for 1 h at room temperature. The primary antibodies include rabbit anti-NLRP3 (PAB38738, Bioswamp, 1:200), rabbit anti-MARK4 (AF0693, Affinity, 1:200), rabbit anti-GFAP (bs-0199R, Bioss, 1:200), rabbit anti-Iba1 (A19776, ABconal, 1:200), rabbit anti-BAX (bs-0127R, Bioss, 1:200). The secondary antibody used is donkey anti-rabbit Alexa Fluor568 (A10042, Invitrogen, 1:1000). Images were captured using a fluorescence microscope (Nikon, Tokyo, Japan), and image analysis was performed using Image J.