Amelogenin

Murine embryos or postnatal pups were used for histology and fluorescence immunohistochemistry. Samples were fixed in 4% PFA, dehydrated, and embedded in paraffin. Sections were cut to 7 μm thickness and standard hematoxylin and eosin staining to assess tissue morphology. Sections that were used for fluorescence immunohistochemistry were rehydrated and treated with 10 mM sodium citrate solution for 20 min at a slow boil for antigen retrieval. These sections were incubated with 10% goat serum–PBST for 30 min at room temperature, followed by overnight incubation at 4 °C with an Ab against one of the following proteins: amelogenin (Santa Cruz; 1:200 dilution), Hmgn2 (Millipore; 1:500 dilution), Ameb (Santa Cruz; 1:200 dilution), Enml (Santa Cruz; 1:200 dilution), Ki67 (Abcam; 1:250 dilution), E-cadherin (BD Bioscience; 1:200 dilution), or Dspp (Santa Cruz; 1:200 dilution). After the incubation, the slides were treated with Alexa-488 (FITC channel) or Alexa-555 (Cy3 channel)–labeled secondary Ab (Invitrogen) at a concentration of 1:500 for 30 min. Each Ab incubation was followed by 3 to 6 PBST washes. Nuclear counterstaining was performed by applying a 4′,6-diamidino-2-phenylindole–containing mounting solution after the final wash (Vector Laboratories). The trichrome staining was carried out as previously described (31 (link)). Samples were stained with azocarmin for 1 h at 50 °C and then stained with aniline to differentiate nuclei. Finally, samples were stained with Orange G and Aniline blue for 2 h.