Immunofluorescence Staining of Brain Tissue

Immunofluorescence staining was performed as described previously [29 (link)]. Briefly, the brain tissue was sampled as described above. The 15-25 μm brain sections were obtained, incubated with phosphate buffered saline (PBS) solution comprising 0.1% Triton X-100 for 30 min, and blocked with PBS solution comprising 5% goat serum (16210064, Gibco, NY, USA) for 30 min. Then, the sections were incubated overnight at 4°C with primary antibodies: IBA1 (ab5076, 1 : 500, Abcam, Cambrige, USA) and CD68 (ab6640, 1 : 200, Abcam, Cambrige, USA). After incubation, the sections were rinsed in PBS for 3 × 5 min washes and incubated for 1 h at 25°C with corresponding secondary antibodies: Alexa Fluor 488 antirabbit IgG (A11034, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 594 antigoat IgG (A11058, Invitrogen, Carlsbad, CA, USA). Lastly, the sections were dyed in DAPI solution (S36939; Invitrogen, Carlsbad, CA, USA) for 15 min at 25°C. The images of all sections were captured blindly using an A1 Si confocal microscope (Nikon, Japan).