Nestin

Staining was performed on cells fixed with ice-cold 4% paraformaldehyde and subsequently permeabilized in blocking solution (1× phosphate-buffered saline pH 7.4, 1% bovine serum albumin, 0.1% Triton X-100). Primary antibodies against FOXG1 (1:100; abcam, Cambridge, United Kingdom), NESTIN (1:300; R&D systems, MN, United States), MAP2 (1:5,000; abcam, Cambridge, United Kingdom), GABA (1:1,000; Sigma, MO, United States), GAD1 (1:100, Millipore, MA, United States) and SST (1:100, Millipore, MA, United States) were used for immunostaining and quantification. Primary antibodies were allowed to bind overnight separately or in appropriate combinations at 4°C. After washing three times in 1×TBS, 0.05% Tween, the secondary antibodies donkey anti-goat IgG AlexaFluor 633, donkey anti-rabbit IgG AlexaFluor 568 or donkey anti-mouse IgG AlexaFluor 488 (1:1,000; ThermoFisher Scientific, Waltham, MA, United States) were applied alone or in appropriate combinations for 1.5 h at room temperature in the dark. Visualization was performed on a Zeiss 510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany) using Zen 2009 imaging software.

We used primary monoclonal antibodies for Sox‐2 (1:50, Biolegend), for CD133 (1:50, Miltenyi Biotec), and for Nestin (5 μg, R&D Systems).

Neuroblastoma cells were treated with cobimetinib (1 μM) and cis-RA (10 μM) alone or in combination for 24 hours. Briefly, the cells were fixed with 4 % paraformaldehyde (Sigma) and permeabilized with 0.05 % Triton X-100 (Sigma). The cells were incubated with antibodies to Nestin (R&D Systems, 1:1000), GFAP (Sigma, 1:1000) and MAP-2 (Sigma, 1:800) for two hours at 37 °C. The cells were then washed with PBS and incubated with fluorescence labelled secondary antibody (Invitrogen, 1:500) at room temperature for one hour. Staining of treated and untreated cells were then visualized by fluorescence microscopy for detection of differentiation markers.

Cells were fixed with 4% (v/v) paraformaldehyde (PFA; Sigma) and stained according to a previously described protocol (Miranda et al., 2015 (link)). MAP2 (Sigma, 1:500), glial fibrillary acidic protein (GFAP, Abcam, 1:200), Synaptophysin (SYN; Abcam, 1:200), ZO-1 (Novex, 1:100), SOX2 (R&D, 1:200), PAX6 (Covance, 1:400), NESTIN (R&D, 1:400), Ki-67 (Abcam, 1:100), HuC/D (Thermo Fischer Scientific, 1:100), activated CASPASE3 (pCASP3, Cell Signaling, 1:400), were used as primary antibodies whereas goat anti-mouse IgG Alexa Fluor–488 or 546 (1:500, Invitrogen), goat anti-rabbit IgG Alexa Fluor–488 or 546 (1:500, Invitrogen) were used as secondary antibodies. Fluorescence images were acquired with Zeiss LSM 710 Confocal Laser Point-Scanning Microscope using 20× and 63× objectives and integrated density were calculated for each channel using ImageJ software. The ratio between integrated density for the marker of interest and nuclear counterstaining with DAPI was calculated for each image. For each staining, the same acquisition settings were applied for all images.

Immunocytochemistry was performed as previously described [23 (link)]. Briefly, cells grown on glass coverslips were fixed with 4% paraformaldehyde and incubated in blocking buffer containing 5% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100 for 30 min. Cells were incubated in primary antibodies at 4°C overnight. Appropriate Alexa Fluor secondary antibodies were used. Primary antibodies include Nestin (1 : 200, R&D systems), Sox1 (1 : 250, Millipore), and Ki-67 (1 : 500, Abcam). Nuclei were identified with DAPI (Sigma). Images were captured using a Zeiss AxioVision microscope with a z-stack split view function. For quantification of Ki-67+ cells, at least 1000 cells were counted for each staining.

Cells were harvested and washed twice with PBS. Cells were fixed with 4% PFA for 15 min, then were incubated with 3% bovine serum albumin (BSA, 82-100-6, Millipore, Burlington, MA, USA) in PBS to detect membrane proteins, or they were incubated with 3% BSA in PBS containing 0.1% saponin (S7900, Sigma-Aldrich) to detect intracellular proteins. Antibodies used for stem cell markers analysis included Nestin (1:200; R&D Systems), CD133 (1:10; Miltenyi Biotec), OCT4 (1:200; Santa Cruz Biotechnology), SOX2 (1:200; AF2018, R&D systems), and NANOG (1:200; ab21624, Abcam). The biotinylated secondary antibodies (VECTOR laboratories) were incubated for 30 min, and then streptavidin PE (BD Bioscience) was added for 10 min. Antibodies used for inflammatory cells analysis were CD45.2-PerCP-Cy5.5 (1:200; 552950, BD Bioscience), CD45-APC (1:200; 559864, BD Bioscience), CD11b-FITC (1:200; 11-0112-82, eBioscience, San Diego, CA, USA), F4/80-PE (1:200; MF48004, Invitrogen) and Ly6G-PE (1:200, 551461, BD Bioscience).
For GFP induction analysis, GFP-N cells (5 × 10⁴ cells) were co-cultured with N-Red or I-Red cells (10⁵ cells) under stem cell culture conditions on days 1−4. The fluorescence intensities were measured by flow cytometry (FACS Verse, BD Bioscience).