Mouse anti nestin

Neurospheres were permeabilized and blocked with a blocking solution consisting of 5% normal goat serum and 0.5% TritonX-100 in PBS at room temperature for 1 h. Neurospheres were incubated with the primary antibodies diluted in the blocking solution overnight at 4°C and then incubated with the Alexa-conjugated secondary antibodies at room temperature for 1 h. Neurospheres were further stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and subsequently mounted onto glass slides with ProLong™ Gold Antifade Mountant (No. P36934, ThermoFisher). Images were acquired on a laser scanning confocal microscope (Leica SP8 TCS) with built-in LAS X software (Leica Biosystem). Images were analyzed with ImageJ software (version 1.45). Primary antibodies and dilutions used in this study were listed in the following: rabbit anti-SOX2 (1:400; #3579; Cell Signaling Technology), mouse anti-Nestin (1:400; #4760; Cell Signaling Technology), rabbit anti-Ki67 (1:100; ab15580; BioLegend), mouse anti-β3-Tubulin (1:200; #4466; Cell Signaling Technology), mouse anti-GFAP (1:1000; ab7260; Abcam), rabbit anti-GFAP (1:5000; ab7260; Abcam), mouse anti-Tuj1 (1:1000; ab78078; Abcam).

Cryostat sections (20 μm) were stained using standard immunohistochemistry. Primary antibodies: rabbit anti-GFAP (1:1000; Dako, Noble Park, VIC, Australia), mouse anti-GFAP (1:1000; Invitrogen, Mulgrave, VIC, Australia), rabbit antidoublecortin (DCX) (1:400; Cell Signaling, Arundel, Qld, Australia), rabbit anti-Pax6 (1:300; Covance), mouse antinestin (1:300; Cell Signaling), mouse anti-β-Tubulin (1:1000; Promega, Alexandria, NSW, Australia); mouse anti-BrdU (1:400; Roche, Hawthorn, VIC, Australia), rat anti-BrdU (1:200; Abcam, Cambridge, MA), mouse anti-HuC/D (1:250; Invitrogen), mouse anti-chondroitin sulfate proteoglycan (CSPG) (clone CS-56) (1:200; Sigma), rat anti mouse-CD11b (1:200; Invitrogen), and mouse anti-Sox2 (1:200, Sigma). Secondary antibodies: Alexa Fluor 488, 568, or 633; 1:1000 (Invitrogen). Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Antigen retrieval was performed by incubation in 2-mol/L HCl for 15 min (BrdU) or 1-mol/L Tris-HCl (pH:8.0) at 90°C for 20 min (HuC/D).