To detect proliferating cells, a pregnant female was injected intraperitoneally with BrdU solution (Invitrogen) 2 hours before harvesting embryos at E13.5. Immunofluorescence with anti-BrdU antibody (Abcam, rat monoclonal, 1:200) was performed following the manufacturer’s protocol, and DAPI was used to stain nuclei. To calculate the percentage of dividing cells, we first counted the total number of nuclei in a defined area from DAPI images, by automated counting using ImageJ plug-in followed by manual confirmation. Then the number of BrdU-positive cells from the same area was counted manually, and this number was divided by the total number of nuclei. The palatal shelf was divided into medial and lateral domains on the coronal sections, by a vertical line drawn from the mid-point of the border that separates the palatal shelf and the rest of the upper jaw (see Figure 5 A-F). Two palatal shelves from each embryo, and three mutant and three control embryos were analyzed. Student’s t-test was used to determine whether the difference was statistically significant (= p < 0.05). To detect apoptotic cells, immunofluorescence with anti-caspase3 antibody (Cell Signaling Technology) was performed as previously described [43 (link)].
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