To begin, add 500 microliters of warm culture gel to a well in a 24-well plate. Quickly transfer the dissected whole incisors isolated from the mouse to the well. Swirl the plate a few times to rinse the incisors.
Add 400 microliters of warm culture gel to a culture dish and to transfer the rinsed incisors to the dish. Orient the incisor to position the cervical loop region at the center of the dish and adjust the tilt of the apical incisor for the desired imaging plane. Once the gel is set, using a pair of fine forceps, remove any gel on top of the region of interest.
Add approximately 150 microliters of warm culture media against the edge of the dish to cover the sample. Place the explant culture at 37 degrees Celsius to allow the tissue to settle. After one hour, turn on the microscope and the two-photon laser.
Secure the culture dish to the stage adapter and place the profusion adapter ring on top. Connect the stage adapter to a temperature controller to maintain the culture at 37 degrees Celsius. Connect the inlet and the outlet of the adapter ring to a microprofusion pump.
Set the profusion speed to 20 and initiate the profusion of the medium over the sample. Place an ACBR on top of the adapter ring and raise the stage so the objective fits through the ACBR to make contact with the culture medium. Turn the laser wavelength to 920 nanometers to visualize GFP.
After locating the sample through an eyepiece, visualize it with a microscope software, then set up Z-step size of four micrometers and a time interval of five minutes for 14 hours. Initiate the time-lapse imaging and save the subsequent files for downstream data analysis. In time-lapse microscopy of the K14-Cre;R26-rtTA;tetO-H2B-GFP cervical loop, the H2B-GFP signals were predominantly observed in the trans-amplifying region of the cervical loop, indicating active cell divisions.
Further, the condensation and alignment of chromosomes at the metaphase plate in mitotic cells followed by their segregation during anaphase and into two daughter cells was observed. Most cell divisions were perpendicular or at an oblique angle relative to the basement membrane with fewer horizontal divisions parallel to the basement membrane. Cell division events were also apparent in K14-Cre;R26-mT/mG cervical loops where all epithelial cell membranes were labeled green.
Mitotic cells were identified by their cell rounding and subsequent cytokinesis. In K14-Cre;R26-mT/mG cervical loops, both horizontal and vertical cell divisions were also observed.
