T8660

Cryosections were blocked in 3% bovine serum albumin (BSA), 10% normal goat serum (NGS), and 0.3% Triton X–100 diluted in PBS for 1 h at room temperature. The sections were incubated overnight at 4°C with primary antibodies diluted in blocking solution. Sections were washed three times with PBS and then incubated with appropriate secondary antibodies diluted in blocking solution for 1 h. The following primary antibodies were used for immunohistochemistry: anti-Pax-6 (1:200; Biolegend #901301), anti-Sox2 (1:100; Santa Cruz #sc-365823), anti-FoxG1 (1:500; Takara #M227), Sox10 (1: 100; Santa Cruz # sc369692), and anti-tubulin III (1:1000; Sigma #T8660).

Matrigel (Corning, Corning, NY, USA) and Neurobasal Medium (Thermo Fisher Scientific) containing B27 supplement (Thermo Fisher Scientific) and GlutaMAX supplement (Thermo Fisher Scientific) were mixed at 1:100. Matrix coating was performed using 300 µL of this mixture, and NS/PCs were incubated for a day. The supernatant was then removed, and NS/PCs were incubated for 14 days in Neurobasal Medium containing 0.05 µL of DAPT (Fujifilm) per mL. Half of the medium was changed every 4 days. Subsequently, 5-FC was administered for 6 days. Cells were fluorescently stained with anti-Nestin (1:200, mouse IgG; Chemicon, Tokyo, Japan; MAB5326), anti-Ki67 (1:500, rabbit IgG; Novocastra, Newcastle, UK; NCL-Ki67p), and anti-beta III tubulin (1:200, mouse IgG2a; Sigma-Aldrich; T8660) antibodies.

Immunocytochemical staining was performed as previously described [33 (link)]. The primary antibodies included dendritic marker microtubule-associated protein 2 (MAP2, chicken, 1 : 4000, NB300-213, Novus), microtubulin marker β-tubulin_III (mouse, 1 : 1000, T8660, Sigma), apoptosis marker cleaved caspase-3 (cl-Casp3, rabbit, 1 : 400, 9664, Cell Signaling), and proliferation marker Ki-67 (rabbit, 1 : 800, AB9260, Millipore). The secondary antibodies included Alexa Fluor 488-conjugated donkey anti-rabbit (1 : 400), Alexa Fluor 568-conjugated donkey anti-mouse (1 : 400), and Alexa Fluor 647-conjugated goat anti-chicken (1 : 200, all from Thermo Fisher Scientific). Cell samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were acquired with an Olympus IX51 microscope equipped with an Olympus DP30BW camera (Olympus Corporation). CellProfiler [34 (link)] and CellProfiler Analyst [35 (link)] were used for image analysis.

To measure axonal mitochondria number, the cells were seeded with approximately 1×10⁴ neurons per well in 96-well plates, fixed and labeled with a mitochondrial marker, anti-ATP5β antibody (1:500, ab14730, Abcam) and anti-βIII-tubulin antibody (1:1000, T8660, Sigma-Aldrich). The cells were then imaged with TiEclipse microscopy (Nikon) using Plan Fluor 60× objective, NA 0.70. The 100 mm axonal length was measured and mitochondria number quantified using ImageJ.

Neurons cultured until day 36 were fixed with 4% paraformaldehyde for 15 min at room temperature and then washed three times with PBS. After an incubation with blocking buffer (PBS containing 5% normal goat serum and 0.3% Triton X-100) for 30 min at room temperature, the cells were incubated overnight at 4°C with primary antibodies at the following dilutions: TDP-43 (rabbit, Proteintech, 10782-2-AP, 1:200) and TUBB3 (mouse, Sigma, T8660, 1:500). The cells were again washed three times with PBS and incubated with secondary antibodies conjugated to Alexa Fluor 488 or 555 (Life Technologies) and Hoechst 33342 (Dojindo Laboratories) for 1 h at room temperature. After three washes with PBS and one wash with distilled water, the samples were mounted on slides and examined using an LSM-710 confocal laser scanning microscope (Carl Zeiss). Line-scan analysis was performed using ImageJ software. The resulting values were normalized to the maximum intensity. For the analysis of TDP-43 localization, TUBB3 staining was used to determine the cell body as the region of interest, and then Pearson’s correlation coefficient was calculated for TDP-43 and Hoechst staining using the Coloc2 plugin.

After incubation with ferumoxytol, cells were plated on laminin‐poly‐L‐ornithine‐coated glass coverslips for 24 hours, fixed with 4% paraformaldehyde (PFA), and Prussian Blue (PB) staining was performed to detect iron‐positive hNPCs, as has been previously described 26. Labeling efficiency was calculated with light microscopy of cells that were PB positive for intracellular iron nanoparticles and expressed as a percentage of positive cells per five high‐power fields. Plated cells were cultured in differentiation medium (Stemline medium [S3194; Sigma Aldrich]), supplemented with 2% B‐27 (17504044; Thermo Fisher) and antimicrobial/bacterial reagent (15240062; Thermo Fisher) for 7 days, fixed; immunofluorescence for glial fibrillary acidic protein (GFAP) (ab7260; Abcam, Cambridge, MA,
http://www.abcam.com; 1/1000) and β‐tubulin III (T8660; Sigma‐Aldrich; 1/1000) was performed to detect differentiation of hNPCs, as has been previously described 25. Slides were stained with fluorophore‐coupled secondary antibodies (GAM488 and GAR594; Thermo Fisher; 1/500) and counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Vectashield H‐1200; Vector Laboratories, Burlingame, CA,
https://vectorlabs.com). Differentiation capacity was performed with fluorescent microscopy on cells that were GFAP or β‐tubulin III positive, expressed as a percentage of cells per five high‐power fields.