Mouse Embryonic Tissue Analysis

Mouse embryos or heads were dissected in phosphate-buffered saline (PBS). Embryos were fixed with 4% paraformaldehyde-PBS solution for 0.5–4 hours. Following fixation, samples were dehydrated through graded ethanol, embedded in paraffin wax and sectioned (7 μm). Standard Hematoxylin and Eosin was used to examine tissue morphology as previous described [157 (link)]. For immunofluorescence (IF) assays, slides were boiled in 10mM sodium citrate solution (pH 6.0) for 20 minutes for antigen retrieval. They were then incubated with 20% goat serum-PBST for 30 min at room temperature, and then with antibodies against Ki67 (Abcam, 1:200), amelogenin (Santa Cruz, 1:200), Lats1 (Cell signaling, 1:200) and pYap (Cell signaling, 1:200) and Beta-galactosidase (Abcam, ab9361, 1:50) at 4 ^oC overnight. The slides were treated with FITC (Alexa-488)- or Texas Red (Alexa-555)-conjugated Secondary antibody for 30 minutes at room temperature for detection (Invitrogen, 1:500). Nuclear counterstaining was performed using DAPI-containing mounting solution.