Time-lapse live imaging.

Materials:

1. Dissecting tools – Fine forceps, #15 feather surgical blade, serrated forceps, spring scissors
2. Dissection media – DMEM/F12 (Thermo 11039047) with 0.5% glucose, warmed to 37C.
3. Culture media A (2X)
   rat serum (Valley Biomedical, AS3061SC, Special Processed)
   1% glucose
   500 µg/ml ascorbic acid 
   2% penicillin–streptomycin
4. Culture media B (1X)
   50% FluoroBrite DMEM (Thermo A1896701)
   50% culture media A
5. Gel for embedding A (2X)
   1.5% low-melting agarose (NuSieve 50080) in DMEM/F12. Heated in microwave to dissolve and kept at 37C until needed.
6. Gel for embedding B (1X)
   50% Culture media A
   50% Gel for embedding A
   Note: mix only when culture media A is warmed to 37C and kept at 37C until needed.
7. Perfusion setup
   We use the Delta T system from Bioptechs (0420-4), that include a micro-perfusion pump, stage and objective heaters, temperature controllers, tubings, and culture dishes.

Experimental procedures:

1. Dissect out the entire mouse mandibular incisor from the surrounding jaw bone in dissection media. Remove periodontal tissues that block the area you wish you image, as the periodontal tissues are rather opaque. A detailed method for dissecting incisors can be found at:
   Chavez, M. G. et al. Isolation and culture of dental epithelial stem cells from the adult mouse incisor. J. Vis. Exp. 87, e51266 (2014).
2. Before embedding in gel, quickly rinse dissected incisors in “Gel for embedding B” and transfer dissected incisors to a Bioptechs Delta T dish.
3. Add just enough fresh “Gel for embedding B” to the dish so the incisor is submerged but not covered on the top by gel. Position the incisor as the gel set so you can clearly image the areas you are interested in. Let gel fully set.
4. Add some “Culture media B” on top of the gel and return the tissue to a 37C cell culture incubator to let tissue recover for at least an hour. 
5. Bring the culture to 2-photon and set up the Delta T perfusion system as company’s instructions, such that “Culture media B”  is flowed (at speed setting 80) over the top of the embedded sample. Samples are maintained at 37 °C, 95% O₂ and 5% CO₂.
6. Images are taken at appropriate wavelength (e.g. 920 nm for GFP) using a Leica Sp8 DIVE two-photon microscope equipped with a 25x 1.1 NA water-immersion lens (or equivalent setup), and at a time interval and length as desired.