Immunofluorescence was performed as previously described by our laboratory (48). Briefly, tissue sections were fixed in 4% polyformaldehyde for 1 min, washed three times with phosphate-buffered saline (PBS), permeabilized with 0.4% Triton X-100 for 30 min, and blocked with goat serum working liquid (Wuhan Boster Biological Technology, Wuhan, China) for 1 hour. The sections were then incubated overnight with mixed primary antibodies at 4°C, washed in PBS to remove unbound primary antibodies, and incubated with secondary antibodies in the dark at RT for 1 hour. The sections were counterstained with 4′,6 diamidino-2-phenylindole (Sigma-Aldrich) for 5 min and washed with PBS. The primary antibodies included rabbit anti-GPR40 (1:50; Santa Cruz, CA), guinea pig anti-MAP2 antibody (1:200; Synaptic Systems, Goettingen, Germany), mouse anti-GFAP antibody (1:100; Proteintech, China), and mouse anti-PSD95 antibody (1:400; Merck Millipore, USA). The fluorophore-conjugated secondary antibodies used were goat anti-guinea pig Alexa Fluor 650 (1:100; Abcam, USA), goat anti-rabbit Alexa Fluor 488 (1:100; Wuhan Boster Biological Technology, China), and goat anti-mouse Alexa Fluor 549 (1:100; Wuhan Boster Biological Technology, China). Images were captured by confocal laser scanning microscopy (Leica, Wetzlar, Germany). The fluorescence intensity was analyzed using Image-Pro Plus 6.0, and colocalization analyses were performed using ZEISS.

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