Mouse anti gfap

Cell climbing slides (diameter, 8 mm) were removed from DMEM/F12 and washed three times with PBS. Slides were fixed with 4% paraformaldehyde (Beijing Solarbio Science and Technology, Ltd.) for 15 min at room temperature. Slides were washed three times in PBS and subsequently blocked with normal goat serum (OriGene Technologies, Inc.) at the working solution provided by the manufacturer for 1 h in a thermostatic water bath at 37°C, prior to incubation with rabbit anti-P2X₇ (cat. no. APR-004-AO; dilution, 1:200; Alomone Labs) and mouse anti-GFAP (cat. no. 644701; dilution, 1:200; BioLegend, Inc.) overnight at 4°C. Slides were then washed three times with PBS, and incubated with fluorescent goat anti-mouse fluorescein isothiocyante (1:200; cat no. ZF-0311; OriGene Technologies, Inc.) and goat anti-rabbit tetramethylrhodamine isothiocyanate secondary antibodies (1:200; cat no. ZF-0313; OriGene Technologies, Inc.) in the dark at 37°C for 1 h. Slides were washed three times with PBS and subsequently stained with DAPI (cat. no. AR1176; dilution, 1:1,000; Wuhan Boster Biological Technology, Ltd.) at 37°C for 60 sec. Slides were washed three times with PBS, sealed with anti-fluorescent quencher (Wuhan Boster Biological Technology, Ltd.) and visualized with a fluorescence inverted microscope.