Ab214185

The expressions of P2X7, NLRP3 and Cleaved caspase-1 in PC12 cells were evaluated via immunofluorescence assay. Immunofluorescence was carried out as previously reported, with slight modifications (McFerran et al., 1998 (link)). In brief, PC12 cells were cultured on 24-well cell plates, fixed by 4% paraformaldehyde for 15 min. Cells were permeabilized with 0.1% Triton × 100 for 20 min and the blocking by 5% BSA for 30 min. The cells were incubated with the antibodies: anti-P2X7 (Proteintech, 28207-1-AP, 1:400), anti-NLRP3 (Abcam, ab214185, 1:400), Cleaved caspase-1 (Thermo Fisher, PA5-99390, 1:400) overnight at 4°C. The next day, the cells were washed three times in PBS followed by further incubation with a fluorescence-conjugated antibody for 2 h at room temperature. The nuclei were counterstained with DAPI. The images were photographed by inverted fluorescence microscopy and quantified by ImageJ software.

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.