Mab2736

Cultured cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min following a pretreatment with 2% PFA in the culture medium for 30 min at room temperature. After being washed, cells were incubated in PBS containing 0.2% Triton X-100 (Sigma-Aldrich) and 10% FBS with primary antibodies at 4°C overnight. Primary antibodies specific to the following proteins were used: CNPase (mouse IgG₁ 1:250; ab6319; Abcam), GFAP (rabbit IgG 1:1000; ab7260; Abcam), human CD63 (mouse IgG₁ 1:200; 556019; Becton Dickinson), MAP2 (mouse IgG₁ 1:200; M1406; Sigma-Aldrich), nestin (mouse IgG_2a, 1:200; MAB2736; R&D Systems, MN, USA), rat CD63 (mouse IgG₁ 1:200; MCA4754GA; AbD Serotec) and SOX2 (rabbit IgG 1:200; ab97959; Abcam). After cells were stained with Alexa Fluor 546 mouse IgG₁ (1:2000; Life Technologies), Alexa Fluor 546 mouse IgG_2a (1:2000; Life Technologies), Alexa Fluor 546 rabbit IgG (1:2000; Life Technologies), Alexa Fluor 488 mouse IgG₁ (1:200; Life Technologies) and Alexa Fluor 488 rabbit IgG (1:200; Life Technologies), they were observed through a BIOREVO BZ-9000 (Keyence, Osaka, Japan) fluorescence microscope. Hoechst 33342 was used to stain cell nuclei.

Newly formed neurospheres and differentiated cells after 48 h of hypoxia were fixed with 4% paraformaldehyde for 10 min at 4°C, washed with PBS and blocked with 10% normal serum for 20 min in PBS that contained 0.5% Triton X-100. The cells were incubated for 24 h at 4°C with primary antibodies against SOX-2 (1:100, MAB2018, R&D Systems, USA), OCT-4 (1:100, MAB1759, R&D Systems, USA), Nanog (1:100, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:100, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:150, MBS462020, MyBiosource, USA), CD15 (1:100, MAB2155, R&D Systems, USA), NESTIN (1:100, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:100, ab130244, Abcam, USA), VEGF (1:100, MAB293, R&D Systems, USA) and HIF1α (1:100, MAB1536, R&D Systems, USA). Neurospheres and cells were washed three times with PBS for 5 min and then incubated at 37°C for 1 h with appropriate fluorophore-labeled secondary antibodies. Images were acquired with a laser scanning confocal microscope (LSM780, ZEISS, Germany).

Cells cultured in hypoxia for 0, 12, 24, 48 and 72 h were collected, subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with antibodies against SOX-2 (1:1000, MAB2018, R&D Systems, USA), OCT-4 (1:1000, MAB1759, R&D Systems, USA), Nanog (1:1000, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:1000, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:1000, MBS462020, MyBiosource, USA), CD15 (1:1000, MAB2155, R&D Systems, USA), NESTIN (1:1000, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:1000, ab130244, Abcam, USA), VEGF (1:1000, MAB293, R&D Systems, USA) and HIF1α (1:1000, MAB1536, R&D Systems, USA). Enhanced chemiluminescence was conducted for visualization.