Nestin

Cells were fixed with 4% paraformaldehyde / 1x PBS. Fixed cells were incubated at 4°C overnight with primary antibody (in 3% BSA/ 1x PBS) with appropriate dilutions: OCT4 (1:500, Santa Cruz), SOX2 (1:500; Stem Cell Technologies), PAX6 (1:300; Covance), Musashi-1 (1:250, Chemicon), Nestin (1:1000, Milipore), FOXO4 (1:500, Cell Signal), β-III Tubulin (1:300, Milipore), MAP2 (1:500, Milipore), GFAP (1:1000, Chemicon and Sigma), ALDH1L1 (1:500, Origene), S100β (1:100, Abcam), and mEM48 (1:50). The secondary antibody was diluted in the blocking buffer according to its best working concentration: Alexa 488 (1:1000; Life Technologies), Alexa 594 (1:1000, Life Technologies), and Cy5 (1:1000, Sigma). The nucleus was visualized with Hoechst 33342 (5 mg/mL). Samples were examined under an epifluorescent scope (Olympus BX51).

hNPCs were fixed, washed, and blocked as previously described13 (link), being followed by a 1 h incubation (at 37 °C) with primary antibodies: nestin (1:200, Millipore, Billerica, MA, USA) to detect neural progenitors, rabbit anti-doublecortin (DCX; 1:500, Abcam, Cambridge, MA, USA) to detect immature neurons, mouse anti-rat microtubule associated protein-2 (MAP-2; 1:500, Sigma) and neuronal nuclei (NeuN; 1:100, Millipore) to detect mature neurons. Secondary antibodies namely, Alexa Fluor 488 goat anti-rabbit immunoglobulin G (IgG, Life Technologies) and Alexa Fluor 594 goat anti-mouse immunoglobulin G (IgG, Life Technologies) were used for 1 h at 37 °C and then 10 min with 4-6-diamidino-2-phenylindole-dihydrochloride (DAPI; Life Technologies). Samples were observed under an Olympus BX-61 Fluorescence Microscope (Olympus, Hamburg, Germany). For this purpose, three coverslips and ten representative fields per condition were chosen and analyzed. In order to normalize the data between the different sets, the results are presented in percentage of cells. This was calculated by counting the cells positive for NeuN/MAP-2/DCX markers, and dividing this value by the total number of cells/field (DAPI-positive cells; n = 3).

After the EdU incorporation assay, cells were washed three times with PBS, and blocked with 100μL/well 3% BSA in PBS for 1 hour at room temperature followed by incubation with the primary antibodies nestin (Millipore, Billerica, MA, 1:500 dilution) and cleaved caspase-3 (CC3, Abcam Inc., Cambridge, MA, 1:500 dilution) overnight at 4°C. nestin is widely used as a marker for stemness while CC3 is a marker of early apoptosis. The wells were then washed 3 times with PBS and species-matched secondary antibody (Alexa Fluor^® 488 or Alexa Fluor^® 555, 1:200, Invitrogen) was applied and incubated for 1 hour at room temperature. The wells were washed 3 times and then incubated with 100μL of 5 μg/mL Hoechst 33342 in PBS for 30 minutes at room temperature, washed three times with PBS, and stored in the dark at 4°C until image acquisition and analysis. NPCs treated with 1μM staurosporine for 6 hours served as a positive control for CC3 experiments. For PI and CC3 experiments, a nuclear/cellular intensity ratio greater than 2 or 2.5 indicated cell death or apoptosis, respectively.

For hydrogel analysis, each sample was fixed in 4% PFA, permeabilized with 0.5% Triton-X and blocked with 2.5% BSA, and then stained against nestin (1:100, Millipore) overnight at 4 °C. After washing, samples were incubated with secondary antibody (1:500, anti-rabbit conjugated Alexafluor 488) mixed with DAPI (1:5000). Imaging was performed on a Zeiss LSM 711 or Zeiss 880 confocal microscope and z-stack images were collected in 4 μm step sizes up to 250 μm for each spheroid. Confocal image analysis of CSC and EC behavior was performed using ImageJ. Cell invasion frequency and distance were assessed using the spheroid periphery as the starting boundary. Individual GBM cells were defined as cells that were not in contact with other GBM cells and/or were physically connected to the spheroid. For spheroid size analysis, the long axis of spheroids was measured using the line measurement tool in ImageJ. Directional orientation of GBM cells and endothelial cells was measured using the angle parameter in ImageJ.

Cells were fixed in 4% (wt/vol) paraformaldehyde for 10 min, permeabilized with 0.2% Triton X‐100 for 5 min and blocked in 3% (vol/vol) goat serum (Dako) or donkey serum (Sigma) for 45 min. They were then incubated in primary antibodies for 45 min followed by secondary antibodies for 30 min (Alexa Fluor dyes, 1:1000, Invitrogen). All antibodies were diluted in the blocking buffer. Nuclei were counterstained with DAPI (Sigma) for 5 min and coverslips were mounted on slides with FluorSave (Merck). All procedures were performed at room temperature. Primary antibodies used in this study were Vimentin (1:100, Millipore), NFIA (1:250, abcam), GFAP (1:500, Dako), GFAP (1:500, Sigma), S100B (1:500, Dako), βIII‐tubulin (1:1000, Sigma), TDP‐43 (1:250, Abnova), NANOG (1:250, R&D Systems), SOX2 (1:250, Millipore), TRA‐1‐60 (1:250, Santa Cruz), OCT3/4 (1:250, Santa Cruz), SOX1 (1:100, R&D Systems), Nestin (1:1000, Millipore), Brachyury (1:100, R&D Systems), EOMES (1:600, abcam), FOXA2 (1:100, R&D Systems), GATA‐4 (1:100, Santa Cruz), SMI32 (1:250, Covance), and Caspase‐3 (1:500, Abcam).
Fluorescent imaging was performed on fields of view containing uniform DAPI staining using either an Axio Observer.Z1 (Zeiss) epifluorescence microscope or an LSM710 confocal microscope (Carl Zeiss). Images were processed and blindly analyzed by using the ImageJ64 (v 1.47) software.