Tcs sp5 cars

We selected six concentrations of Taxol and designed seven experimental groups as follows: 0 nmol/L (control), 1 nmol/L, 2 nmol/L, 3.5 nmol/L, 5 nmol/L, 7 nmol/L, and 10 nmol/L treatments. 5 × 10⁴ cells/well were seeded evenly on a circular PDL/Laminin-coated coverslip of 12 mm diameter, and the NSC adherent culture medium comprising DMEM (Invitrogen) +10% FBS (Invitrogen) + GlutaMAX (Gibco) + NEAA (Gibco) + Penicillin-Streptomycin (Gibco) was added. Cells were grown and allowed to adhere in a CO₂ incubator at 37°C for 24 hours. Subsequently, the culture medium in the seven groups was replaced by the NSC differentiation medium with Taxol at different concentrations. The cells were then allowed to differentiate for 7 days at 37°C in a CO₂ incubator. After 7 days, the immunofluorescence staining assay was performed using anti-Tuj1 (βIII-tubulin, 1 : 500, Sigma), anti-Map2 (1 : 500, Sigma), and anti-GFAP (1 : 800, Sigma) antibodies. The cells were incubated with the primary antibodies overnight at 4°C followed by incubation with secondary antibodies at 26°C for 2 hours. The cells were scanned using a confocal microscope (Leica TCS SP5 CARS).

The rats were anesthetized. After perfusion, the spinal cord tissues were dissected and fixed in 4% paraformaldehyde for 72 hours, followed by immersion in 20% and 30% sucrose solutions for tissue dehydration, successively for 24 hours each. The frozen tissue was cut into 20 μm thick sections (Leica CM1950).
Tissue sections were HE stained following the kit protocol (Beyotime C0105M). The images were captured using a digital slide scanner (3DHISTECH Pannoramic).
For the immunofluorescence assay, the tissue sections were treated with 0.3% PBST and blocked in 10% BSA for 1 hour. The sections were incubated with primary antibodies, including anti-GFAP (1 : 800, Sigma) and anti-Tuj1 (βIII-tubulin, 1 : 500, Sigma) overnight at 4°C followed by secondary antibody incubation at 26°C for 2 hours. The tissue sections were scanned using a confocal microscope (Leica TCS SP5 CARS).