U3750

Organotypic hippocampal slice cultures were prepared from P5 C57BL/6 wild-type mice of both sex according to.³⁷ (link) In brief, mice were decapitated and the hippocampus was extracted and sliced into coronal sections of 300 μm thickness. The slices were placed on small pieces of Polytetrafluoroethylene (PTFE) membrane, pore size 0.45 μm (Millipore, # FHLC01300) and cultured on cell culture insert of 0.4 μm pore size (Millipore, # PICM03050) placed in a 6-well plate and then incubated at 37°C with 5% CO₂ for 18 to 23 days in vitro (DIV). The inhibitor mix containing Ara-C (Sigma-Aldrich, #C6645), Uridine (Sigma-Aldrich, #U3750) and 5-Fluoro-2′-desoxyuridine (Sigma-Aldrich #F0503), was added to a final concentration of 3 μM on the third day and the medium was exchanged three times per week.

DRG were collected from 2–4‐week‐old Sprague‐Dawley (SD) rats. Bilateral DRG were cultured in vitro in a 35 mm culture dish and studied under a microscope. Next, neuronal fibers connected to the DRG were removed and sensory neurons were isolated from the DRG by sequential digestion using papain and type I collagenase (1 mg mL⁻¹) for 3 h, as reported previously.^[66] Thereafter, 5000 cells mL⁻¹ of these isolated cells were cultured in a Neurobasal medium (A358290, Thermo Fisher Scientific) containing 2% B27 (A3582801, Thermo Fisher Scientific), 0.3% L‐glutamine (25030081, Thermo Fisher Scientific), 100 ng mL⁻¹ NGF (N2513, Sigma‐Aldrich), 1% PSN, 10⁻⁵ M fluorodeoxyuridine (F0503, Sigma‐Aldrich), and 10⁻⁵ M uridine (U3750, Sigma‐Aldrich). These cells were cultured in a six‐well plate coated with poly D‐lysine hydrobromide (50 ng mL⁻¹; Life Technologies). The following day, the medium was replaced with one containing different treatment reagents; subsequently, the medium was refreshed every two days. SiRNA transfection (siRNA ID#: s155625, Thermo Fisher Scientific) (Sequence (5′→3′): Sense: CCGUGGGUGUCUAACUUCAtt; Antisense: UGAAGUUAGACACCCACGGct.) was applied in vitro to knockdown the expression of Sema5b.