Anti idh1

Protein extracts from rat xenograft tissue were resolved on 4–12% BisTris gel (NP0323, Lifetech, Gent, Belgium) and transferred on PVDF membranes (Invitrolon PVDF, Lifetech, # LC2005). Blocking of the membrane was done with 2% milk in Tris-buffered saline solution with 0.1% triton X-100. Protein detection was done with the following primary antibodies: Anti-malectin C-terminal (1/6000, SIGMA, Diegem, Belgium, # SAB4200245) and anti-IDH1 (1/1000, DiaNova, Hamburg, Germany, # DIA-W09). Normalization of the signal were done with actin (detection with anti-actin, clone C4 (1/10000, millipore, # MAB1501)). Secondary antibodies (peroxidase-conjugated) were respectively: anti-Rabbit IgG (H&L) (Jackson ImmunoResearch, Suffolk, UK, #111–036-003), anti-Rat-IgG (H+L) (DiaNova, # 112–035-062), ECL Anti Mouse IgG (GE healthcare #NA931V/AG). Signal detection was performed with Super Signal West Femto Maximum Sensitivity Substrate-(Thermo SC # 34095) according to manufacturer's instructions. Image acquisition was done with an ImageQuant LAS4010 imaging station and signal intensities were quantified with Image Quant TL software (GE Healthcare, Belgium).

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).