To begin, place the shaved, anesthetized mouse on a warming pad. Pinch the skin with tweezers near the fourth nipple and cut the raised skin with sharp scissors to make a midline incision of approximately one centimeter. Make circular incisions around the gland in both cranial and caudal directions, and carefully peel the skin away from the abdomen.
Remove any exogenous blood promptly with physiological saline to avoid subsequent loss of optical resolution. To minimize blood loss, seal any prominent blood vessels with a handheld cauterizer. Then gently tease the surface layer with fine forceps to remove superficial connective and adipose tissue.
Keep the exposed gland moist with physiological saline. If required, treat the gland with exogenous agents, for example, pharmacological agents or organelle specific dyes by bathing the exposed glands during surgery. Protect the abdominal wall with a semi-transparent, flexible thermoplastic film.
Carefully position the mouse with the abdominal side down on an inverted microscope stage such that the skin flap extends onto the central cover glass. Protect the exposed areas with a thin layer of gel. Tape the rear leg and tail to the stage.
Then place a custom made notched spacer prepared from three taped cotton sticks between the skin flap and the body wall to cushion the gland from motion artifacts caused by breathing and heartbeat. Securely tape a plastic cover at either end of the stage opening to prevent the skin flap from sliding during the imaging. Insert a subcutaneous indwelling catheter under the dorsal skin attached to a tube, syringe, and pump.
Using conventional fluorescence microscopy, confirm that the skin flap is stable with adequate blood flow. Perform conventional confocal microscopy of the EGFP cyto or EGFP membrane mouse labeled with BODIPY 665676 and two photon microscopy of the EGFP membrane mouse labeled with monodansylpentane and tdTomato membrane mouse labeled with monodansylpentane. In EGFP cyto mice, the cytoplasm of secretory epithelial cells was distinctly labeled by EGFP.
At the same time, lipid droplets stained with BODIPY 665676 appeared as round fluorescent bodies, especially towards the apical surface. Time-lapse analysis showed BODIPY 665676 stained lipid droplets moving from basal to apical regions at variable speeds and fusing with each other in transit. In EGFP membrane mice, EGFP highlighted the plasma membranes of capillary and secretory epithelial cells and BODIPY stained lipid droplets were visible throughout the cytoplasm.
Two photon microscopy of the mammary glands of either EGFP membrane or tdTomato membrane mice allowed a more comprehensive survey of gland morphology. Collagen fibers at the surface are visualized in the monodansylpentane channel through second harmonic generation. Further into the parenchyma, the secretory epithelium is revealed with basal, lateral, and apical plasma membranes and monodansylpentane stained lipid droplets.
The central lumen becomes obvious further into the alveolus. The red tdTomato fluorophore was especially useful for labeling the capillary endothelium, unlike EGFP in the EGFP cyto mice.
