2.5. Immunofluorescence analysis

For immunocytochemical staining, primary cells were rinsed with 0.1 M phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for 20 min. Cell cultures on the cell slides were blocked with 5% bovine serum albumin (BSA) in PBST (0.3% Triton X-100) and then incubated with the primary antibody at 4 °C overnight. The cells were washed with PBS and then exposed to Alexa Fluor 555 goat anti-mouse (1:1000, Invitrogen, A21422, CA, USA) or Alexa Flour 488 goat anti-rabbit (1:1000, Invitrogen, A11008) for 1 h at room temperature. After they were rinsed with PBS, the cell slides were sealed with Antifade Mountant (ProLong™ Diamond Antifade Mountant, Invitrogen, {"type":"entrez-protein","attrs":{"text":"P36965","term_id":"544168","term_text":"P36965"}}P36965) and observed under an Olympus scanning microscope (Olympus BX51, Japan).

Immunofluorescence in brain slices was detected as described in our previous study [18]. The brains were dehydrated with 20% sucrose-PBS and then 30% sucrose-PBS for 3 d respectively after they were fixed in 4% paraformaldehyde. The brain tissues were sliced into serial brain sections with a thickness of 25 μm. The sections were blocked with 10% FBS in PBST (0.3% Triton X-100) for 1 h, followed by incubation overnight with a primary antibody at 4 °C. The sections were then washed with PBS and incubated with Alexa Fluor 488 conjugated goat anti-rabbit (1:1000, Invitrogen, A11008) or Alexa Fluor 555 goat anti-mouse (1:1000, Invitrogen, A21422) for 1 h at room temperature. The sections were ultimately rinsed with PBS and then mounted onto adhesive slides. Fluorescently labeled sections were visualized with a scanning microscope (Olympus BX51, Japan). The primary antibodies used for immunofluorescent staining were as follows: mouse anti-kir6.2 antibody (1:200, Santa Cruz, sc-390104, CA, USA), rabbit anti-GFAP antibody (1:1000, Abcam, ab7206, Cambridge, UK), rabbit anti-MAP2 antibody (1:200, Proteintech, 17490-1-AP, Wuhan, China), rabbit anti-PM20D1 antibody (1:200, Biorbyt, orb555691, Cambridge, UK), and mouse anti-Tau (1:200, Santa Cruz, sc-32274),.