Goat anti mouse af488

Coronal paraffin sections (8 µm) were blocked with 2% BSA and 0.1% saponin, incubated for 2 hours with primary antibodies rabbit anti-Iba1 (1∶200) (Wako Chemicals, Osaka, Japan) and mouse anti-GFAP (1∶100) (Acris antibodies) followed by incubation with secondary antibodies goat anti-rabbit AF594 and goat anti-mouse AF488 (both Invitrogen). Nuclei were counterstained with DAPI (Invitrogen) and mounted with FluoroSave reagent (Calbiochem). Fluorescent images were captured with an AxioCam MRm (Carl Zeiss, Sliedrecht, The Netherlands) on an Axio Observer Microscope with Axiovision Rel 4.6 (Carl Zeiss).

HeLa were cells seeded on CellView slides (Greiner Bio-One). They were infected with VACV EGFP-A5 for 30 min at RT. The inoculant was then replaced with the indicated compounds in the text, figures, and figure legends. After 20 h at 37°C, cells were washed and fixed with 4% EM grade FA in PBS. They were permeabilized and blocked simultaneously in 0.5% Triton-X 1000 in 5% BSA in PBS. Anti-I3 antibody (generously provided by Jakomine Krijnse Locker, Institute Pasteur) was used at 1:1,000. All secondary antibodies (goat anti-mouse-AF488 and goat anti-rabbit-AF647, Invitrogen) were used at 1:1,000. Primary I3 antibody was added for 60 min at RT, followed by a wash and 60 min RT staining with secondary antibody and DAPI. Images were captured using a 100× oil immersion objective (NA 1.45) on a VT-iSIM microscope (Visitech, Nikon Eclipse TI), using 488 and 640 nm laser frequencies for excitation.

Briefly, PFA-fixed mNSCs from the co-culture assay were blocked with 5% BSA and 0.1% saponin for 30 minutes followed by incubation for 1 hour at room temperature with primary antibodies: mouse anti-nestin (1∶200) (BD Biosciences, Breda, The Netherlands), rabbit anti-Olig2 (1∶400) (Millipore), mouse anti-GFAP (1∶100) (Acris antibodies, Herford, Germany) or rabbit-anti βIII-Tubulin (1∶1000) (Abcam antibodies, Cambridge, UK). Secondary antibodies goat anti-mouse AF488 or goat anti-rabbit AF594 (Invitrogen, Paisley, UK) were incubated for one hour at room temperature. Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI; Invitrogen) and mounted with FluoroSave reagent (Calbiochem, Nottingham, UK). Fluorescent images were taken with an AxioCam MRm (Carl Zeiss, Sliedrecht, The Netherlands) on an Axio Observer Microscope with Axiovision Rel 4.6 software (Carl Zeiss).

Pooled HUVECs (human umbilical vein endothelial cells) were obtained from Lonza and expanded over two passages in collagen 1 (from rat tail, BD) coated flasks in complete EGM-2 medium supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a 5% CO₂–3% O₂ humidified chamber. HUVECs were transfected twice at 24 h-interval with 30 nM siRNA (Dharmacon smartpool ON-TARGET plus Thermo Scientific; Non-targeting siRNA #1, CCM2 ref. L-014728-01) and Lipofectamine RNAi max (Life Technologies, ref. 13778-150) according to the manufacturer’s instructions at 37 °C in a regular 5% CO₂ in a humidified chamber. The day after transfection, HUVECs were seeded at 1.5 × 10⁵ cells in 24-well plates on coverslips coated with 10 μg/ml fibronectin (F0895, Sigma Aldrich) and incubated for 48 h total in complete EBM-2 medium supplemented with increasing concentrations of RA (Sigma Aldrich) with one renewal after 24 h. Cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100, and incubated with anti-beta-catenin mouse monoclonal antibody (6F9 clone, Sigma Aldrich). After rinsing, coverslips were incubated in Goat anti-Mouse AF 488 (Invitrogen, 1:1000) and phalloidin conjugates with TRITC (Sigma, 1:2000). The coverslips were mounted in Mowiol/DAPI solution and imaged on an epifluorescent Axio Imager microscope (Zeiss) at 63× magnification.