Rabbit anti nestin

Cell populations were dissociated with Pasteur pipettes and plated onto Matrigel GFR-coated glass coverslips (BD-Italia) to perform the immunostaining. Cells were fixed in 4% paraformaldehyde and permeablized with 0.2% Triton-100 and subsequently processed for immunolabeling. The following primary antibodies (Ab) were used: mouse anti-MAP2 (a/b), and rabbit anti-Nestin (Millipore). The secondary Ab used were: fluorescein isothiocyante (FITC) affinity purified goat anti-rabbit and Tetramethyl rhodamine isothiocyanate (TRITC) affinity purified donkey anti-mouse (Chemicon). Nuclei were stained with Hoechst 33258 diluted in PBS (0,2 μg/ml; SIGMA). Cells were photographed at 400X magnification using a fluorescent microscope (Olympus microscope OLYMPUS Bx5 with Spot CCD Camera). A quantitative cell analysis was performed by counting for each antibody used almost 100 cells/field of five fields from each of three independent experiments.

Human iPSC-derived motor neuron cultures were plated on optical-bottom 96-well plates (Thermo, # 165305) and subsequently fixed in 4% paraformaldehyde for 15 minutes. Cells were blocked in 5% normal donkey serum with 0.1% Triton X-100 in PBS and incubated with primary antibodies for 1 h at room temperature or overnight at 4°C. Cells were then rinsed and incubated in species-specific AF488, AF594, or AF647-conjugated secondary antibodies followed by Hoechst 33258 (0.5 μg/mL; Sigma) to counterstain nuclei. Cells were imaged using Molecular Devices ImageExpress Micro high-content imaging system or using Leica microscopes (Fuller et al., 2015 (link)) (Figure 1B). Primary antibodies used were as follows: mouse anti-SMI32 (Covance, 1:1,000); mouse anti-TuJ1 (β3-tubulin) (Sigma; 1:1,000-1:2,000); rabbit anti-GFAP (Dako; 1:1000); mouse anti-Map2a/b (Sigma; 1:1000); rabbit anti-nestin (Millipore; 1:2000), Islet-1 Antibody (R&D AF1837; 1:250) and Nkx-6.1 (DSHB F55A10-s; 1:100).

Cells were washed with PBS, fixed with 4% PFA, treated with glycine 0.2 M, permeabilized with Triton X-100 at 0.2% and then blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich) and 5% goat serum. Slides were mounted on Fluoromount-G (SouthernBiotech, 0100-01) and stained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; ThermoFisher Scientific, D1306). Immunolabeling was performed at 4°C overnight with the following primary antibodies: rabbit anti-MAP2 (1/25, Cell Signaling Technology, 4542), rabbit anti-Nestin (1/200, Millipore, AB5922), mouse anti-Neurofilament-M and Neurofilament-H (1/50, Millipore, MAB1592), mouse anti-CD14 (1/5, Immunotech, IOM2) and mouse anti-Tubulin (1/100, Zymed Laboratories, Invitrogen, 13-8000). Immunodetection was performed using species and subclass specific Alexa Fluor-405, Alexa Fluor-488 or Alexa Fluor-647 conjugated secondary antibodies (1/200, Life Technology). Antibodies were diluted in PBS containing 0.1% saponin and 0.3% BSA. Actin was stained with rhodamine phalloidin (1/200, Molecular Probes, Invitrogen, R415). Images were visualized under a wide-field microscope (Leica DMI 6000) equipped with a Micro MAX-1300YHS camera using an HCX PL APO 60X oil objective (Princeton Instruments). Images were acquired using Metamorph Software (Version 7.1.3; Molecular Devices).