Musashi

ESCs and MSCs were fixed with Bouin’s solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 4 °C and stored. Fixed cells were blocked, permeabilized and incubated with the primary antibodies overnight in TCT buffer (0.25% Carrageenan and 0.1% Tween-20 in Tris-HCl buffer pH 7.8). The primary antibodies used were: Sox2 (1:100, Abcam, Cambridge, UK), E-Cadherin (1:15, BD Biosciences, San Jose, CA, USA), CD44 (1:100, Abcam), βI Integrin (1:100, Abcam), Musashi (1:200, Abcam) and Vimentin (1:100, Abcam). After rinsing, appropriate secondary antibodies conjugated to Alexa fluorophores 488 or 598 (Molecular Probes, Invitrogen, Carlsbad, CA, USA) were diluted at 1:500 in TCT solution and incubated for 1 hr at room temperature. After rinsing, cells were mounted in Vectashield hardset mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector laboratories) to counterstain nuclei. Images were acquired with a Leica DMI 6000B microscope.

TS formation from human GBM specimens followed previous methods (Kong et al. 2013b (link)), and their expression of stemness markers, CD133, nestin, musashi, and podoplanin (Abcam, Cambridge, UK), was tested by immunocytochemistry. Neuroglial differentiation in GBM TSs was evaluated by monitoring the expression of GFAP (Dako, Carpinteria, CA, USA), MBP, NeuN, and TUBB3 (Chemicon, Temecula, CA, USA).