Tie inverted fluorescence microscope

Mice were anesthetized by an IP injection of a mixture of ketamine (100 mg kg⁻¹) and xylazine (20 mg kg⁻¹). The small intestine was surgically externalized, and the epithelium was exposed via a small incision in an area devoid of intestinal content. During the procedure, the epithelial tissue was constantly moistened by applying saline. The anesthetized mouse was placed on the microscopic stage and covered with a heated pad (37 °C) to maintain body temperature. Fixable fluorescent dextran conjugates of 3 or 2000 kDa in size (ThermoFisher D-3305 or D7137) were injected directly into the intestinal lumen via the incision, and the externalized epithelium was then positioned on a coverslip mounted on the stage above the objective and immobilized using custom-made holders. The blood flow was assessed visually by using the eyepiece and only regions close to blood vessels were imaged. The microscope used was a NIKON TiE inverted fluorescence microscope equipped with a Yokogawa CSU-21 spinning disc head and an Andor DU-897 camera. NIKON Elements software was used for image analysis.

COS7 cells were imaged using a Nikon TiE inverted fluorescence microscope, outfitted with a spinning disk confocal scan head (Yokogawa), 100 × 1.49 N.A. objective, and Andor DU-888 EM-CCD. Coverslip-bottom dishes were placed in a stage top incubator with CO2 concentration maintained at 5%, and temperature at 37C under humidified conditions for the duration of the imaging experiments. Multichannel images were acquired at 500 ms exposures per channel, every 5 seconds for an average of 10 min per dataset. Acquisition of all datasets was managed through NIS-Elements software.