Mouse anti human nestin

Immunohistochemistry was performed as previously described [44] (link). Briefly, injected animals were perfused at different time points with 4% paraformaldehyde (PFA). The brains were removed and immersed in 4% PFA overnight at 4°C and then equilibrated in 30% sucrose. Entire brains were processed in sagittal 40 microns microtome sections. Sections which were positive with GFP-labelled cells were blocked with 5% donkey serum, permeabilized with 0.25% Triton X in TBS for 30 minutes before application of primary antibodies of rabbit anti-GFAP (Dako, Glostrup, Denmark), anti-PDGFRα (Millipore), goat anti-doublecortin (Santa Cruz, CA, USA), mouse anti-human nestin (Millipore) and anti-human nuclei (Millipore) at dilutions of 1∶100 to 1∶500 for overnight incubation at 4°C. Incubations with secondary antibody (1∶500) of 647 donkey anti-rabbit or 647 donkey anti-goat with 555 donkey anti-mouse for one hour at room temperature were performed, followed by a five minute staining with DAPI (Millipore), before sections were mounted on slides with mounting medium. The staining was viewed using Zeiss LSM 710 confocal system (Carl Zeiss Pte Ltd, Singapore) at 63X magnification.

Cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 15 min for intracellular staining. Cells were then blocked with 3% bovine serum albumin (BSA) (Sigma) in PBS for 1 h at room temperature and incubated with primary antibodies diluted in 1% (w/v) BSA in PBS overnight at 4°C. Cells were washed with PBS three times and stained with secondary antibodies for 1 h at room temperature in the dark. The following primary and secondary antibodies were used: mouse anti-human OCT4 (AbD Serotec, UK), rabbit anti-human NANOG (Millipore), Alexa Fluor 488 anti-human TRA-1-60 antibody (BioLegend), mouse anti-human TRA-1-81 (Millipore), mouse anti-human Stage-Specific Embryonic Antigen-4 (SSEA-4) (Millipore), mouse anti-human NESTIN (Millipore), rabbit anti-human TUJ1 (Covance), mouse anti-human α-fetoprotein (Calbiochem), mouse anti-human actin (muscle) clone HHF35 (AbD Serotec), mouse anti-human PAX6 (Millipore), Alexa Fluor 488 γoat anti-mouse IgG (H+L), Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 568 goat anti-mouse IgG (H+L) (Life Technologies). Nuclei were stained with prolong gold DAPI with antifade (Invitrogen). Cells were visualized with an Eclipse TE2000 fluorescent microscope (Nikon, Japan).

NPCs were seeded onto a Matrigel-coated black plate, clear-bottom 96-well plate (Corning 3603), across a range of cell densities. At 24 h after plating, NPCs were fixed in 4% paraformaldehyde in PBS at 4 °C for 10 min. NPCs were permeabilized at room temperature for 15 min in 1.0% Triton in PBS, blocked in 5% donkey serum with 0.1% Triton at room temperature for 30 min and mouse anti-human NESTIN (Millipore), 1:200 and 200 units per ml phalloidin-568 (Life Technologies) were incubated overnight at 4 °C. Secondary antibodies were Alexa donkey 488 anti-mouse (Life Technologies) at 1:200. To visualize nuclei, slides were stained with 0.5 μg ml⁻¹ DAPI (4′,6-diamidino-2-phenylindole). High-content 96-well imaging (× 20 air objective) of NESTIN (red), F-ACTIN (phalloidin, green) and DAPI-stained NPCs was performed using an ImageXpress Ultra (confocal). Images were captured (4 images per well, 12 wells per NPC line, 16 total NPC lines (from six controls and four SZ patients)) and cell segmentation analysis was completed using MetaXpress (Molecular Devices). Data were exported using AcuityXpress (Molecular Devices). Density-matched wells were manually identified; total area of F-ACTIN-positive cells was compared.