Gt10312

Fresh-frozen human brain tissues were acclimated to room temperature and then exposed to post-fixation by 4% paraformaldehyde. Following a 10 min wash in buffer (TBS containing 0.1% Triton-X), tissues were exposed to protein block (5% goat serum, 0.1% Triton-X 100, 1% BSA; in TBS). Tissues were incubated with rabbit anti-PBR (ab109497; Abcam, 1:3000), and one of the following: mouse anti-Iba1 (GT10312; Invitrogen, 1:500), mouse anti-CD68 (M0814; Agilent, 1:500), mouse anti-GFAP (G3898; Millipore Sigma, 1:500) or antibody diluent overnight at 4°C and then washed with TBS buffer 3 × 5 min. For detection, sections were incubated with a goat anti-rabbit secondary antibody conjugated to Alexa-Fluor-568 (Invitrogen) and a goat anti-mouse secondary antibody conjugated to Alexa-Fluor-488 (Invitrogen) at room temperature for 60 min. Following washing and 10 min incubation with DAPI, slides were washed with distilled water and coverslipped. Slides were imaged using an Olympus VS200 (Olympus Corporation).

Immunofluorescence staining was performed as described previously [30 (link)]. Briefly, the frozen sections were incubated with primary antibody at 4°C overnight: mouse anti-Iba1 (1 : 200, #GT10312, Invitrogen, USA), mouse anti-GFAP (1 : 200, #3670S, Cell Signaling Technology (CST), USA), mouse anti-NeuN (1 : 200, #ab104224, Abcam, USA), rabbit anti-LRP1 (1 : 200, #64099S, CST, USA), rabbit anti-TXNIP (1 : 200, #14715S, CST, USA), and rabbit anti-NLRP3 (1 : 200, #15101S, CST, USA). After washing with phosphate-buffered saline (PBS), the sections were incubated with the corresponding secondary antibody (Cy3-labeled goat anti-rabbit IgG, FITC-labeled goat anti-mouse IgG) (1 : 200, #S0011, #0007, Affinity, USA) at room temperature for 1 h, followed by DAPI (#C0060, Solarbio, China) staining. Finally, the sections were visualized, and images were captured using a fluorescence microscope.