Embryonic Tissue Preparation and Analysis

Mice were euthanized at predetermined stage and embryos were collected in cold PBS, fixed in 4% PFA overnight, washed in PBS for 3 times and processed to 100% methanol (for whole mount in situ hybridization), or paraffin for section (for histology staining and section in situ hybridization). Histology staining and in situ hybridization procedures were performed as previously described (Liu et al., 2013 (link), 2015 (link)). In situ probe templates were amplified by PCR from total cDNA sample of E13.5 wildtype forelimbs. Antisense RNA probes were synthesized from templates using T7 RNA polymerase (Promega catalog# P2075). Immunofluorescent staining was performed as previously described (Xu et al., 2014 (link)). The primary antibodies used include anti-Sox9 (Santa Cruz, catalog# sc-20095, 1:25 dilution) and anti-FLAG antibody (Sigma, catalog# F1804, 1:50 dilution). The secondary antibodies were Alexa Fluor 568-conjugated goat anti-mouse IgG (H + L) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) [(Thermo Fisher Scientific, Waltham, MA, United States), catalog# A11004 and A27034, 1:500 dilution].