Embryos were harvested at desired developmental stages (E13.5 to E18.5, noon of vaginal plug date was counted as day E0.5), fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C, dehydrated through graded alcohols, embedded in paraffin, sectioned at 7μm thickness, and stained with alcian blue (A), hematoxylin (H) and eosin (E) for histology analysis. At least four pairs of control and mutant embryos were analyzed for each developmental stage.
Whole mount and section in situ hybridization assays were performed using established protocols (Lan and Jiang 2009 (link); Lan et al. 2001 (link); Liu et al. 2013 (link); Zhang et al. 1999 (link)).
Immunofluorescent staining was performed using paraffin or frozen sections following standard protocols (Lan et al. 2016 (link); Xu et al. 2016 (link); Zhou et al. 2011 (link)). Antibodies used include rabbit anti-Giantin (GOLGB1, Abcam ab24586; 1:300), mouse anti-GM130 (BD610823; 1:500), rabbit anti-GOLGA5 (Sigma-Aldrich HPA000992; 1:300).
Whole mount and section in situ hybridization assays were performed using established protocols (Lan and Jiang 2009 (link); Lan et al. 2001 (link); Liu et al. 2013 (link); Zhang et al. 1999 (link)).
Immunofluorescent staining was performed using paraffin or frozen sections following standard protocols (Lan et al. 2016 (link); Xu et al. 2016 (link); Zhou et al. 2011 (link)). Antibodies used include rabbit anti-Giantin (GOLGB1, Abcam ab24586; 1:300), mouse anti-GM130 (BD610823; 1:500), rabbit anti-GOLGA5 (Sigma-Aldrich HPA000992; 1:300).
