Embryonic Mouse Head Explant Culture

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.