Anti cleaved caspase 1

After different treatments, the cells were incubated with RIPA lysis buffer containing a protease inhibitor cocktail on ice for 10 min. Cell lysates were then harvested by scraping, followed by brief sonication and centrifugation at 12,000 × g for 10 min at 4 °C. The protein concentration was determined via the BCA assay. Equal amounts of protein were separated by SDS/PAGE and the bands were electroblotted onto PVDF membranes. The membranes were blocked with 5% non-fat milk dissolved in TBST and then incubated with appropriate primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-NLRP3 (1:1000, Abcam), anti-Gasdermin D-N-terminal (1:1000, Invitrogen), anti-caspase-1 (1:1000, Invitrogen), anti-cleaved caspase-1 (1:1000, Invitrogen), anti-GAPDH (1:10000, Abcam), α-Fodrin (1:1000, Cell Signaling Technology), anti-ATP5A1 (1:1000, Invitrogen), Calpastatin (1:500, ABclonal), and Flag (1:1000, Abcam). The membranes were then incubated with the following horseradish peroxidase-conjugated secondary antibodies: HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG purchased from Proteintech (1:5000, America). The immunofluorescent bands were then visualized and quantified using an ECL chemiluminescence kit (Absin, Shanghai, China) and a chemiluminescence–western blotting detection system (Tanon, Shanghai, China).

Following treatment, cells were fixed with 4% paraformaldehyde for 10 min before permeabilization with 0.1% Triton X-100 in PBS for 10 min. Cells were then blocked for 1 h in normal goat or horse serum before overnight incubation at 4 °C with their respective primary antibodies; anti-NLRP3 (1:500; Abcam, Cambridge, UK), anti-cleaved caspase-1 (1:100, Invitrogen, Carlsbad, USA), anti-NLRP1 (1:500; Novus Biologicals, Littleton, CO, USA), or mouse anti-RPE65 (1:1000; Abcam, Cambridge, UK). Two 10 min washes in PBS followed, after which cells were incubated at room temperature for 2 h with their respective secondary antibodies; donkey anti-rabbit Alexa-488 (1:500; Abcam, UK), donkey anti-mouse Alexa-488 (1:500; Abcam, Cambridge, UK), or donkey anti-goat Cy3 (1:500; Invitrogen, Carlsbad, USA). Cells were then washed twice in PBS for 10 min. Cell nuclei were counterstained with DAPI (1:1000; Sigma-Aldrich, St Louis, MO, USA), and slides were mounted using a CitifluorTM antifade reagent.

Immunohistochemistry was carried out as previously reported with minor modifications (Aricioglu et al., 2019 (link)). Brain tissue was fixed with 4% paraformaldehyde for 24 h. Dehydrated, embedded in paraffin, and cut into slices of 5 µm thickness. After a series of operations, including deparaffinization, antigen repair and blocking, sections were incubated overnight with primary antibodies: anti-BDNF (Abcam, ab108319, 1:200), anti-P2X7 (Proteintech, 28207-1-AP, 1:50), anti-NLRP3 (Abcam, ab214185, 1:200) and anti-Cleaved caspase-1 (Thermo Fisher, PA5-99390, 1:200) at 4°C, followed by secondary antibodies incubation. Then, the slices were colored with diaminobenzidine (DAB). Finally, after dehydration and drying, sections were observed using a light microscope. The density values are analyzed by Image pro-plus software.