Immunofluorescence and in situ Hybridization

Tissue was embedded in paraffin as previously described (May et al., 2015 (link)). Immunofluorescence was performed either on paraffin-embedded tissue or on whole-mount dissected embryonic salivary glands and explant cultures (Gaete et al., 2015 (link)). Primary antibodies and dilutions were used as follows: anti-Sox9 1:300 (AB5535, Millipore); anti-BrdU 1:500 (ab6326, Abcam), anti-Sox2 1:200 (#2748, Cell Signaling Technology); anti-Mist1 1:50 (sc-98771, Santa Cruz Biotechnology), for which signal was amplified with the TSA kit (PerkinElmer); anti-laminin 1:300 (L9393, Sigma); anti-K5 1:300 (119-13621, Cambridge Bioscience); anti-Sox10 1:100 (sc-365692, Santa Cruz Biotechnology) using the TSA kit; anti-cleaved caspase 3 1:200 (#9661, Cell Signaling Technology). In situ hybridisation was performed as previously described (Gaete et al., 2015 (link)). Plasmids for probe generation have been described previously: Spry1 (Minowada et al., 1999 (link)), Fgf10 (Bellusci et al., 1997 (link)), Myb (Matalová et al., 2011 (link)), Etv5 (Hippenmeyer et al., 2002 (link)) and Col2a1 (Ng et al., 1997 (link)).