Immunofluorescence Staining of GFP and CD45

Immunofluorescence stainings for GFP and CD45 co-expressing cells were performed on 20 μm thick fresh frozen brain sections as previously described [22 (link)]. Cryostat cut sections were air-dried, rinsed in TBS for 10 min before blocked with 10% fetal calf serum in TBS + 0.5% Triton for 30 min at RT. Next, sections were incubated with rabbit anti-mouse GFP (ab290, Abcam) overnight at 4 °C. The following day the sections were rinsed for 10 min in TBS + 0.5% Triton before incubation with the cross-absorbed species-specific secondary antibody Alexa Flour 488 goat anti-rabbit (#A11070, Invitrogen) diluted 1:200 for 2 h. Next, the sections were rinsed in TBS for 10 min before incubation for 2 h with PE-conjugated rat anti-mouse CD45 (#553081 BD Biosciences). After a 2 × 10 min rinse in TBS the sections were transferred to TBS containing 40,6-diamidino-2-phenylindole (DAPI) (D1306, Invitrogen), rinsed in water, and mounted in gelvatol. Respective isotype controls were devoid of signal.