Anti hunu

Cells grown on chamber slides (BD) were fixed (paraformaldehyde 4% in PBS, 15 min). Every following step was performed in PBS containing 1% BSA (Sigma). Cells were permeabilised (0.1% Triton X-100, 25 min) and blocked with 5% normal goat serum (NGS, 15 min). Primary antibody incubation was performed at 4°C overnight, dilution 1 : 200. Primary antibodies, anti-HuNu (Millipore), anti-cTnT (Abcam), anti-Mef2c (New England Biolabs), anti-connexin 43 (Abcam), and anti-alpha-sarcomeric actinin (Abcam), were washed and samples were incubated with secondary antibodies (1 : 500) for 30 minutes at RT. Secondary antibodies used were as follows: anti-rabbit-Alexa488, anti-mouse-Alexa594, and anti-rabbit-Alexa555 (Invitrogen) and anti-mouse Alexa555 (Cell Signaling).
Stained specimens were kept in mounting media (PermaFluor, ThermoScientific) and images were acquired using a fluorescent microscope (EVOS, Life Technologies) or spinning disk confocal microscope (Quorum Zeiss AxioVert, SickKids Imaging Facility, Toronto). Images were quantified using Volocity™ (Perkin-Elmer) imaging software.

Mouse brains were embedded in Tissue-Tek O.C.T. (Sakura Finetek, Alphen aan den Rijn, The Netherlands) and snap frozen in isopentane (Sigma-Aldrich) cooled on dry ice. Snap-frozen tissue was sectioned at 6 μm thickness on a cryostat (Leica CM3050S, Leica Microsystems, Wetzlar, Germany). Subsequent washes were done with TBS-Tween20 (Sigma-Aldrich) wash buffer, 3×3 min, all steps were performed at room temperature. The primary antibodies used were: anti-ANGPT2 (1:100, Abcam), anti-Msi1 (1:500, Abcam), anti-NG2 (1:100, Abcam), anti-Sox2 (1:100, Abcam), anti-VEGF (1:20, Abcam), anti-Vimentin (1:100, Abcam), anti-PDGFRα (1:100, Cell Signaling Technology, Beverly, MA, USA), anti-GFAP (1:500, Dako, Glostrup, Denmark), anti-Tubulin β3 (1:100, Millipore), anti-HuNu (1:100, Millipore), anti-FGF2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IDH1 (Dianova, Hamburg, Germany) and anti-POU3F2 (SC-2895, Santa Cruz). The secondary antibodies used were: FITC-conjugated goat anti-rabbit (1:200, Southern Biotech), FITC-conjugated goat anti-mouse (1:200, Southern Biotech), TXRD-conjugated goat anti-mouse (1:100, Southern Biotech).