Mouse anti iba1

Immunohistochemical staining methods were described previously.¹¹, ²¹ The samples were incubated with rabbit anti‐S1PR3 (1:100, Bioss, China), mouse anti‐NeuN (1:100, Bioss, China), mouse anti‐GFAP (1:100, Bioss, China), mouse anti‐CD31 (1:100, Abcam, USA), and mouse anti‐Iba‐1 (1:100, Bioss, China) antibodies (Abs). ZO‐1 quantitative colocalization analysis was performed in a previous study,²² and two independent observers performed the counts. The coverage coefficients of ZO‐1/CD31, S1PR3/CD31 were calculated using ImageJ.

Sections were deparaffinized in xylene and rehydrated through graded alcohol series. After that, sections were washed three times in phosphate-buffered saline (PBS) for 5 min each, then incubated with 5% goat serum for 1 h at 37 °C. Sections were incubated with the rabbit anti-PPAR-γ, anti-iNOS (1:100, Proteintech, Wuhan, China), rabbit anti-Arg1 (1:200, Bioss, Beijing, China), rabbit anti-Aβ_1–42, anti-P-Tau (1:200, Abcam, Waltham, MA, USA), mouse anti-Iba1 and anti-F4/80 (1:200, Bioss, Beijing, China) overnight at 4 °C. The sections were incubated with Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (1:200, Proteintech, Wuhan, China) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Bioss, Beijing, China) at 37 °C for 2 h in a dark room after being washed 3 times with PBS. In the final step, the sections were rinsed 3 more times before being stained for 5 min with DAPI (Biyuntian, Shanghai, China). The sections were imaged using Pannoramic MIDI (3DHISTECH, Budapest, Hungary).

Cells were fixed with 4% paraformaldehyde for 10 min and then washed in phosphate-buffered saline (PBS). After being blocked with 5% bovine serum albumin containing 0.5% Triton X-100 for 1 h at 37°C, the microglia were incubated with the following primary antibodies at 4°C overnight: mouse anti-Iba1 (1:200, Bioss, Beijing, China), rabbit anti-iNOS, anti-Arg1, anti-IL-1β, anti-TNF-α, anti-IL-4, anti-IL-6 and anti-IL-10 (1:200, Bioss). After being washed in PBS three times, the cells were incubated with the following secondary antibodies at 37°C for 1 h: Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Bioss) or Alexa Fluor Cy3-conjugated goat anti-mouse IgG (1:200, Bioss). After being washed in PBS three times, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Biyuntian, China) for 5 min. The cells were then washed in PBS and observed under a 7266-fluorescence microscope (Leica, Japan).