Before placing the anesthetized mouse on the live imaging insert installed on the inverted microscope stage, thread the isoflurane tubing with the attached nose cone through the corner tubing bracket on the insert. After plugging the heat plate into the controller containing an attached anal thermometer probe and switching it on, transfer the anesthetized mouse from the induction chamber onto the heat plate. Then secure the nose cone onto the mouse.
Insert the anal probe into the animal and adjust the controller to maintain a stable body temperature of about 36 degrees Celsius. Cover the properly-positioned mouse with a plastic cling wrap to trap body heat and elevate the body temperature. Position the mouse such that its head aligns with the cover slip disc and immobilize its ear onto the center of the glass cover slip using a metal ear clip or tape.
After completing imaging, select the Low Flow option on the electronic vaporizer to cease isoflurane delivery. Transfer the mouse to a heating pad until it becomes ambulatory. After removing the cover slip disc, wipe it clean with lens paper and lens solution.
Store it properly to avoid scratches for reuse. Next, detach the anesthetic tubing from the insert bracket and unscrew the insert from the microscope stage. The acquired Z-stacks of fluorescently-labeled live mouse ear tissue showed minimal drift in the x, y and z axes over time, validating the custom 3D printed live imaging inserts.
The time points from a three hour time lapse movie of the mCherry positive mouse ear epidermal cells of an adult male mouse revealed a single z plane remaining in focus over the extended time. Similar observations were made from another three-hour time lapse movie of GFP positive ear dermal fibroblasts of an adult female mouse.
