Mouse anti cytokeratin 14

Indirect immunofluorescence staining was carried out on paraffin sections using the following primary antibodies: rabbit anti-PP2Ac (1:100, Cell Signaling); mouse anti-cytokeratin-14 (1:50, Santa Cruz, Dallas, CA, USA); mouse anti-pan-cytokeratin (1:50, AE13, Santa Cruz, Dallas, CA, USA); mouse anti-trichohyalin antibody (1:100,AE15, Abcam, Cambridge, UK); Rabbit anti-Ki67 (1:100, Abcam, Cambridge, UK); secondary antibody kit: Rabbit SP detection kits (KIT-9706) (Maxim, FuZhou, China), mouse SP detection kits(KIT-9701) (Maxim). Quantification of fluorescence was performed by a blinded observer using the ImageJ software (National Institute of Mental Health, Bethesda, MD, USA) and depicted as percent of relative expression.

After 3-D co-culturing, the Matrigel^TM/cell complex was embedded in OCT compound (Sakura Finetek, Torrance, CA, USA) and then cryosectioned at a thickness of 10 μm and stored at −80 °C.
For immunofluorescence, the cryosections were retrieved from −80 °C and incubated in 3% goat serum, 0.1% BSA and 0.1% Triton (blocking solution) for 1 hr at room temperature. The sections were incubated with primary rabbit anti-amelogenin antibody (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4 °C. After being washed thoroughly, the sections were incubated with a second primary antibody, mouse anti-cytokeratin 14 (1:200, Santa Cruz Biotechnology, Inc.), for 1 hr at room temperature. After being washed thoroughly, the sections were incubated with both FITC-conjugated anti-rabbit (1:160, Santa Cruz Biotechnology, Inc.) and TRITC-conjugated anti-mouse IgG (1:200) (Sigma-Aldrich, St. Louis, MO, USA) secondary antibodies for 1 hr. Nuclei were counterstained with 0.5 μg/mL Hoechst 33342 (Invitrogen Corporation) in the dark for 5 min. After mounting, the tissue sections were imaged using a Nikon Eclipse 300 fluorescence microscope (Compix Inc., Sewickley, PA, USA).