βiii tubulin

The mice were anesthetized and perfused with PBS followed by 4% paraformaldehyde (PFA) at indicated times. After postfixed in 4% PFA for 6 h, the brains were dehydrated in sucrose (10%, 20%, and 30%) and embedded in Tissue-Tek OCT compound (Sakura® Finetek USA). Then, the brains were cut into 25 μm thick sections for histological staining. The brain sections and stem cell coverslips were fixed with 4% PFA for 20 min, followed by blocking with a solution containing 0.3% Triton, 3% goat serum, and 1% BSA. Subsequently, samples were incubated with primary antibodies against BRCA1 (1 : 200, Abcam, UK), GFP (1 : 500, Cell Signaling Technology, USA), BrdU (1 : 200, Abcam, UK), Nestin (1 : 200, Sigma-Aldrich, USA), βIII-tubulin (1 : 400, Cell Signaling Technology, USA), GFAP (1 : 400, Cell Signaling Technology, USA), and CNPase (1 : 400, Abcam, UK) overnight at 4°C. After being washed with PBS for three times, the samples were incubated with appropriate secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, USA). Images were captured by FLUOVIEW FV1000 confocal microscopy (Olympus, Japan).

Cells and tissues were fixed with 4% (w/v) paraformaldehyde for 30 min at room temperature and then permeabilized in 0.1% (v/v) Triton X-100 for 30 min at room temperature. Nonspecific antibody binding was blocked by incubating in 5% (w/v) bovine serum albumin for 30 min. Samples were incubated overnight with the following primary antibodies: Nestin (10 μg/mL; Sigma), Sox2, Oct4 (10 and 2.5 μg/mL, respectively; Abcam, UK), β-III tubulin, Map2 (both 5 μg/mL; Cell Signalling, UK), and GFAP (2 μg/mL; Life Technologies). On the following day, complementary Alexa Fluor 488- and 594-conjugated secondary antibodies (both 4 μg/mL; Life Technologies) were applied. Glass coverslips were mounted using mounting media supplemented with DAPI stain (VectorLabs) and preparations imaged with Deltavision imaging software with X/Y/Z multiple position recording (Applied Precision).

Cells were lysed in RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche) and the phosphatase inhibitors NaF and Na₃VO₄ (Sigma). Cell lysate (40 μg) was separated by SDS-PAGE and blotted onto nitrocellulose. The blots were incubated with the indicated antibodies and developed by enhanced chemiluminescence (GE Healthcare). Antibodies against the following proteins were used: phosphatase and tensin homolog (PTEN), phospho-Akt (Ser473), Akt, phospho-S6 ribosomal protein (Ser235/236), S6 ribosomal protein, FoxO1, FoxO3a, FoxO4, Musashi, p21, GFAP, βIII-tubulin, cleaved caspase 3 (Cell Signaling); actin, Mdm2 (Santa Cruz); p53, Sox2 (R&D Systems); Nestin (Millipore). HRP-conjugated secondary antibodies were purchased from Dianova. Quantification of signals was performed using Image Quant TL (Amersham Bioscience).

Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Samples were blocked with 0.1% Triton X-100 (Sigma-Aldrich) and 5% donkey serum in PBS for 30 min and were subsequently incubated with primary antibodies against NANOG (marker for pluripotency; R&D Systems), βIII-tubulin (neuronal marker; Cell Signaling Technology), MAP2 (neuronal marker; Merck-Millipore) and tyrosine hydroxylase (TH, marker for dopaminergic lineage; Merck-Millipore) at 4°C overnight, followed by incubation with appropriate secondary antibodies (Molecular Probes, Thermo Fisher Scientific) conjugated to AlexaFluor 488 (green) or 546 (red), for at least 1 h at room temperature. Coverslips were mounted with ProLong Gold antifade reagent with DAPI (Cell Signaling Technology) and images were acquired using a Leica TCS-SP5II confocal microscope (LEICA Microsystems).

Primary cortical neurons were prepared from C57BL/6 mouse brains according to the established protocol [10 (link)]. Briefly, cortices were isolated from P0 pups and digested with 100 U of papain for 30 min at 37 °C and mechanically disrupted by pipetting several times in neurobasal medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% B27 (Invitrogen) and a 1% glutamine/glutamax mixture (1:3) (Invitrogen). The dissociated cortical neurons were seeded in poly-D-lysine-coated 24-well plates (0.5 × 10⁴ cells/well) or 8-well glass slide chambers (2 × 10³ cells/well) for cAMP or neurite outgrowth analysis, respectively.
Neurons were stained for β-III tubulin (1:200, cat# 5568, Cell signaling, Danvers, MA, USA) and images were acquired using a Zeiss LSM 700 confocal laser-scanning microscope. The neurite length was measured using NIH Image J software (National Institutes of Health, Bethesda, MD, USA) as we described earlier [10 (link)].

hHF-MSC-derived iPSCs were harvested by collagenase type IV. After settling, the supernatant was aspirated and the MEF medium was replaced to remove the MEF. hHF-MSC-derived iPSCs were transferred to petri dishes in the MEF medium. After an 8 days floating culture, embryoid bodies were transferred to gelatin-coated plates and were then incubated for another 16 days. After the incubation, the cells were fixed with 4% paraformaldehyde in PBS and then incubated in PBS containing 5% normal goat serum (Maixin Biotech, Fuzhou, China), 1% bovine serum albumin (BSA, Biotopped, China), and 0.2% Triton X-100. The primary antibodies were as follows: anti-alpha smooth muscle actin (α-SMA, R&D, USA), anti-alpha fetoprotein polyclonal antibody (AFP, R&D, USA), and anti-Nestin (R&D, USA). vimentin(Cell Signaling, USA), desmin (Cell Signaling, USA), βIII-tubulin (Cell Signaling, USA). The secondary antibodies were Alexa 555-labeled anti-mouse IgG (1:1000, Cell Signaling, USA). Nuclei were stained with 1 mg/ml Hoechst 33342 (Invitrogen, USA).