Microslicer zero 1

Following normoxia or CIH exposure, rats were anesthetized with isoflurane (2–3%), perfused transcardially with cold-modified artificial cerebrospinal fluid (modified aCSF in mM: 103 sucrose, 63 NaCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 3 KCl, 0.5 CaCl₂, 2.5 MgCl₂, 2 MgSO₄, 10 d-glucose) equilibrated with Carbogen (95% O₂, 5% CO₂), and their hindbrains were isolated. Coronal slices containing caudal NTS (250 µM thick) were cut in a MicroSlicer Zero 1 (Ted Pella Inc.) using a sapphire knife (Ted Pella Inc.). For protective recovery, slices were transferred to an incubation chamber containing prewarmed (34°C) aCSF composed of (in mM) 126 NaCl, 3 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 2 CaCl₂, 2 MgSO₄, and 10 d-glucose (300 mosmol/L) for ∼30 min. Slices remained in the recovery chamber at room temperature for another 1 h before proceeding with patch-clamp recordings. Incubation solutions were saturated with carbogen at least 10 min before use to ensure stable pH buffering (pH 7.3) and adequate oxygenation.

Embryonic mouse heads were dissected and collected in complete BGJb Medium: BGJb Medium (Thermo Fisher Scientific, 12591038) with 10% Fetal Bovine Serum (12662029, Thermo Fisher scientific), 0.1 mg/ml L-ascorbic acid (A4403, Sigma-Aldrich), and 1% Penicillin-Streptomycin (15140148, Thermo Fisher Scientific). Mouse heads were embedded on ice in a mixture of preheated 20% gelatin (G2500-500G, MilliporeSigma) in BGJB culture medium and sectioned to 300 μm slices using MicroSlicer Zero 1 (10111, Ted Pella). These slices were placed on cell culture inserts (PICM0RG50, Sigma Aldrich) which were inserted into 6-well culture plates and cultured at 37 °C in a 5% CO₂ incubator in complete BGJb Medium. For bead implantation, Affi-Gel blue agarose beads (1537302, BioRad) were soaked in 1 μg/μl FGF18 (100–28, Peprotech) or BSA for one hour at 37 °C (Xu et al., 2016 (link)) and then implanted into the soft palatal shelves of E14 Osr2^Cre;Tgfbr1^fl/fl head slices. The samples were collected 3 days after bead implantation.