Pa579740

The paraffin sections of 4 μm brain tissue were routinely dewaxed to water, microwave heated with sodium citrate antigen repair solution for 10 min for antigen repair, washed with PBS, and sealed with 10% sheep serum (Solebo, SL038) at room temperature for 1 h. The blocking buffer was vacuumed, and diluted IBA1 primary antibody (1: 400, Wako, 019-19741) and rabbit anti-NLRP3 (PA579740, 1:1,000, Thermo Fisher, USA). Then the sample was incubated overnight at 4°C, and rinsed with 1× PBS for three times prior to the incubation with the second antibodies including Goat anti-rabbit IgG-H&L (Alexa Fluor^® 594)(1:1000, Abcam, ab150080) and Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) (1:1000, Abcam, ab150165) at room temperature in dark for 1 h. Each sample was incubated with DAPI (Solebo, C0065) at dark for 10 min. After staining, the staining solution was removed and rinsed with 1× PBS for three times. The slides were sealed quenchable sealing tablets and observed under Fluorescence microscope.

Mice were perfused with 4% PFA. Eyeballs were removed and fixed with 4% PFA for 24 h. Frozen sections of 10 μm thickness were embedded in optimal cutting temperature compound (OCT, Tissue-Tek, Sakura Finetek, USA). TUNEL staining was performed using in situ Cell Death Detection Kit, Fluorescein (11684795910, Roche, USA). For immunofluorescence staining, the primary antibodies used were as follows: rabbit anti-NLRP3 (PA579740, 1:1,000, Thermo Fisher, USA), mouse anti-Caspase-1 (sc-56036, 1:100, Santa Cruz, USA), and Rabbit anti-IL-1β (12703, 1:100, Cell Signaling Technology, USA). The secondary antibodies used were goat anti-rabbit (A-11034, 1:1,000, Thermo Fisher, USA) and goat anti-mouse (A-11032, 1:1,000, Thermo Fisher, USA). After that, sections were imaged using a fluorescence microscope (TS100, Nikon, Japan) and analyzed using ImageJ.