Goat anti sox10

For embryonic tissues, freshly dissected intestines were fixed 1 hour at RT with 4% PFA in PBS. Alternatively, whole embryos were fixed overnight at 4°C and the intestines dissected afterwards. For adult tissues, whole intestines were fixed in 4% PFA overnight at 4°C, cut longitudinally along the mesentery and washed in PBS. The outermost muscle layers were then stripped from the mucosa/submucosa. Fixed tissues were dehydrated in methanol. After rehydration, the intestines were incubated 2 hours at RT in blocking solution (10% fetal bovine serum, 0.1% Triton-X100 in PBS). Tissues were incubated with primary antibodies overnight at 4°C. The antibodies used were: mouse anti-βIII-Tubulin (1:200; Abcam ab78078), rabbit anti-S100β (1:500; Dako Z0311), goat anti-Sox10 (1:100, Santa Cruz Biotech. sc-17342) and rabbit anti-Ki67 (1:1000; Abcam ab15580). Secondary antibodies Alexa Fluor 594- or Alexa Fluor 647-conjugated anti-goat, -mouse or -rabbit (1:500, Jackson Immunoresearch) were incubated for 2 hours at RT and counterstained with DAPI. All antibodies were diluted with blocking solution. For the TUNEL assay, tissues were permeabilized 20 min at 37°C in 0.3% Triton-X100, 0.1% sodium citrate in PBS 1x, then stained in a 1:9 mix of enzyme solution:label solution from the in situ cell death detection kit, TMR red (Roche Applied Science 12156792910) 1h at 37°C.

Cells or gut explants were fixed in PBS containing 4% PFA for 15 min, permeabilized for 5 min in PBS containing 0.25% TritonX-100 and incubated in PBS containing 10% BSA and 0.01% sodium azide for 30 min at RT. The cells were then incubated with the following primary antibodies diluted in PBS containing 3% BSA and 0.01% sodium azide: goat anti-NRP1 (1/500), mouse anti-TuJ1 (1/500), guinea-pig anti-synapsin 1 (Synaptic Systems, 1/700) or goat anti-SOX10 (Santa Cruz, 1/300) for 16 h at RT. For F-actin labeling, cells were incubated for 1 h at 37 °C with Alexa Fluor 568-phalloidin (1/10,000, Thermo Fisher). After washing, cells were incubated for 1h30 min at RT with the appropriate Alexa 488- or Cy3-conjugated secondary antibodies, and then for 5 min in DAPI 1/5,000 and mounted with ProLong Gold Antifade Reagents.