Anti gfap

The primary antibodies used were: anti-filaggrin (1:500; Abcam, catalog number # ab24584), anti-fibronectin (1:200; R&D Systems, catalog number # AF19181), anti-GFAP (1:400; R&D Systems, catalog number # AF2594), anti-βIII tubulin (1:200; R&D Systems, catalog number # MAB1195), anti-vimentin (1:200; R&D Systems, catalog number # MAB2105), anti-nestin (1:200; R&D Systems, catalog number # MAB2736), anti-CNPase (1:400; Chemicon, catalog number # MAB326), CD34-FITC (Pharmingen, San Jose, CA catalog number # 553733) and anti-p63 (1:500, AbCAM, catalog number # ab53039). The secondary antibodies were: Texas Red anti-sheep (1:1,000 catalog number # TI-6000), FITC anti-sheep (1:500 catalog number # F7634), FITC anti-rabbit (1:1,000 catalog number # FI-1000), anti-sheep HRP (1:1,000 catalog number # A16-147), anti-rabbit HRP (1:1,000 catalog number # PI-1000) all from Vector Laboratories, USA. The fluorophore-conjugated antibodies for flow cytometry were: CD34-PE catalog number # 551387, CD31-APC catalog number #561814, CD45-PE catalog number # 553081, CD44-PE catalog number # 561860, CD90-FITC catalog number # 554894, CD29-PE catalog number #562801, CD105-PE catalog number # 562759 diluted 1:500, all from BD Biosciences (San Jose, CA, USA).

Neurospheres of T4213 CD133⁺ cells were collected by centrifugation at 200 × g for 10 minutes and washed with PBS, followed by immersion in Tissue-Tek OCT compound (Sakura). Frozen sections were prepared. T4213 CD133^– cells on culture slides were washed with PBS and fixed with 3% paraformaldehyde. The sections and culture slides were stained with anti-SOX2 (1:200, Abcam) and anti-GFAP (1:100, R&D System) antibodies, and visualized by using Alexa Fluor 488- and 568- conjugated IgGs and Alexa Fluor 633-labeled phalloidin (Invitrogen). Cells were examined by confocal scanning using a TCS-SP microscope (Leica, Heidelberg, Germany).