Alexa fluor 594 conjugate anti mouse secondary antibody

An original method was applied to label proteins in the glioma neurospheres. Neurospheres were placed into cell insert (Millipore, US) and fixed by 0.4% Paraformaldehyde solution (Solarbio, China). Then, the neurospheres were washed three times in PBS, and incubated with primary antibodies (See Supplementary Table 1) overnight at 4°C. Alexa-Fluor 488 conjugate anti-rabbit secondary antibody (1:5000, Cell signaling, US) and Alexa-Fluor 594 conjugate anti-mouse secondary antibody (1:5000, Life technologies, US) were used for fluorescent double-staining. Serum medium cultured CD133+ cells were stained through Alexa-Fluor 594 conjugate phalloidin (1:200, Life technologies, US) for 20 minutes at 37°C to exhibit actin cytoskeleton. DAPI solution (Solarbio, China) was employed to label cell nucleus. Images were observed and captured by Perkinelmer UltraVIEW VOX confocal microscope (Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China).

An original method was applied to label proteins in the glioma tumorspheres. Tumorspheres were placed into a cell insert (Millipore, US) and fixed with 0.4% paraformaldehyde solution (Solarbio, China). Then, the tumorspheres were washed three times with PBS, and incubated with primary antibodies overnight at 4 °C. Alexa-Fluor 488 conjugated anti-rabbit secondary antibody (1:1000, Life Technologies, USA) and Alexa-Fluor 594 conjugate anti-mouse secondary antibody (1,1000, Life Technologies, USA) were used for fluorescent double-staining. DAPI solution (Solarbio, China) was employed to label cell nuclei.