Pe conjugated rat anti mouse cd45

For identifying cellular localization of IFN-β, we used IFN-β/YFP reporter mice. Tissue sections (16 µm) were rinsed in PBS containing 0.2 % Triton X-100 (PBST). The sections were incubated in blocking solution containing PBST and 3 % BSA, and stained either with rabbit anti-GFP antibody (ab6556; Abcam), PE-conjugated rat anti-mouse CD45 (BD Biosciences), Cy3-conjugated anti-GFAP or anti-Mac-1/CD11b (MCA711, Serotec, Oxford, UK). After 3 washes in PBST, sections were incubated with biotinylated goat anti-rabbit (Abcam), Alexa-569 goat anti-rat or streptavidin-HRP. GFP signal was amplified with TSA fluorescein kits (PerkinElmer) according to the manufacturer’s instructions. Sections were incubated with DAPI and mounted using gelvatol [17 (link)].
Images were acquired using an Olympus DP71 digital camera mounted on an Olympus BX51 microscope (Olympus, Ballerup, Denmark) and with Olympus FV1000MPE Confocal and Multiphoton Laser Scanning Microscope, Danish Molecular Biomedical Imaging Center (DaMBIC), University of Southern Denmark. Images were combined using Adobe Photoshop CS3 (Adobe Systems Denmark A/S, Copenhagen, Denmark) to visualize double-labeled cells.

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.