Chunk #21 — Methods — Reagents and antibodies

Source

Establishment of human iPSC-based models for the study and targeting of glioma initiating cells.

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The following antibodies were used at the specified concentrations for immunostainings: sex-determining region Y-box 2 (SOX2; AB5603, 1:500; Chemicon); Oct-3/4 (sc-5279, 1:500; Santa Cruz Biotechnology); NANOG (EB06860, 1:500; Everest Biotech); NANOG (21624, 1:400, abcam); anti-phospho-Akt Ser473 (#9271, 1:50; Cell Signaling); anti-phospho-ERK1/2 Thr202/Tyr204 (#9101, 1:50; Cell Signaling); neuron-specific class III β-tubulin (#MRB-435p and MMS-435p, TUJ-1; 1:500; Covance); microtubule-associated protein 2 (#AB5622, MAP2; 1:500; Millipore); PAX6 (PRB-278P; 1:1000; Covance), glial fibrillary acidic protein (#Z0334, GFAP; 1:500; Dako); α1-fetoprotein (A0008, AFP; 1:400; Dako); calponin (M3556, 1:500; Dako); Ki67 (ab16667; 1:500; abcam); NESTIN (MAB5326, 1:500; Millipore); human nuclear antigen (#MAB1281, HUNU; 1:250, Millipore); O4 (MAB345, 1:200; Milipore), PECAM-1 (#sc-1506, CD31; 1:50; Santa Cruz Biotechnology); 4,6-diamidino-2-phenylindole (DAPI) (5 mg ml−1) 1:2,000 (D1306, Invitrogen, Carlsbad, CA); and Alexa Fluor 488 goat anti-mouse (1:1,000, Invitrogen), Alexa Fluor 488 donkey anti-goat (1:1,000, Invitrogen), Alexa Fluor 488 donkey anti-rabbit (1:1,000, Invitrogen) Alexa Fluor 568 donkey anti-mouse (1:1,000, Invitrogen), Alexa Fluor 568 donkey anti-rabbit (1:1000, Invitrogen) and Alexa Fluor 647 chicken anti-rabbit (1:1,000, Invitrogen).