Mandibular slice cultures were performed as previously described (Wells et al., 2013 (link); Li et al., 2016 (link)). For the bead experiment, two types of beads were used to help distinguish between the control and treated conditions. For the Fgf10-treated explants, heparin beads (Sigma, 100-200 mesh) were incubated overnight at 4°C with 100 μg/ml Fgf10 (R&D Systems). For the control, Affi-Gel blue beads (Bio-Rad,153-7302) were treated with 0.5% BSA. For inhibiting Fgf receptor signalling or the Erk pathway, explant cultures were treated with 2.5 μM SU5402 (Merck) or 5 μM U0126 (Cell Signaling Technology), respectively, made up in DMSO. Control cultures were treated with equivalent concentrations of DMSO (0.25% DMSO for the SU5402 and 0.5% DMSO for the U0126 experiment). For the collagenase treatment, whole E12.5 submandibular glands were dissected and treated for 2 days with 1 μg/ml collagenase, Type II (Thermo Fisher Scientific) and HBSS-treated glands were used as a control. Spooner ratios were calculated as the number of buds at the end of culture divided by the number of buds at the start of culture.
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