Glial fibrillary acidic protein (gfap)

The prefrontal cortices of mice were analyzed via immunofluorescent assays. Coronal sections (5-μm thickness) were prepared and immersed in 4% PFA for 30 min, followed by three washes in TBST. After blocking non-specific binding via 1.5% BSA for 1 h, sections were then incubated with corresponding primary antibodies overnight at 4 °C, as follows: GFAP (1:50; Proteintech, Wuhan, Hubei, China) and P-ERK1/2 (1:100; Beyotime, Shanghai, China); GFAP and GLUT3 (1:100; Santa Cruz, Dallas, Texas, USA); NeuN (1:50; Proteintech, Wuhan, Hubei, China); P-ERK1/2; and GFAP and T-ERK1/2 (1:100; Santa Cruz, Dallas, Texas, USA). After washing three times with TBST, the secondary antibody mix with FITC-conjugated goat anti-mouse IgG (1:200; Bioss, Beijing, China) and Cy3-conjugated goat anti-rabbit IgG (1:200; Bioss, Beijing, China) was added to cover the tissue and was incubated at room temperature for 1 h in the dark. Sections were then incubated with DAPI at room temperature for 5 min. Fluorescent images were obtained, and image analysis was applied to quantify immunoreactive signals.

Primary retinal pericytes were extracted from fetal bovine eyeballs and cultured in dulbecco's modified eagle medium (DMEM) containing 20% foetal bovine serum (FBS), as described previously [18 (link)]. All cells were cultured at 37°C in 5% CO₂, and the media were changed every 2–3 days. We use primary pericytes from passages 3–5 for this study. α-smooth muscle actin (α-SMA, Abcam, Cambridge, UK) and nerve/glial antigen 2 (NG2, Santa Cruz, Dallas, TX, USA) were used as positive stains to assess primary bovine retinal pericytes; factor VIII (Santa Cruz) and glial fibrillary acidic protein (Santa Cruz) were used as negative stains. The cells were immobilized with paraformaldehyde (4%) for half an hour, then infiltrated with Triton X-100 (0.5%) for 20 mins. The primary antibodies were incubated at 4°C overnight; the secondary antibodies combined with FITC (ZSGB-BIO, Beijing, China) were incubated at 37°C for 1 h. Finally, the cell nuclei were stained by 4′6-diamino-2-phenylindole (Beyotime, Shanghai, China), and the cells were detected by fluorescence microscopy (Life Technologies, EVOS FL Auto). Subsequently, the pericytes were pretreated with or without chloroquine (CQ, 10 μM, Sigma, St. Louis, MO, USA) for 2 h and then exposed to AGE-BSA (Biovision, San Francisco, CA, USA) or Control-BSA (Biovision) for 24 h.