Imaging Fixture Supported Mouse Liver Staging for Multiphoton Microscopy Based Vascular Visualization: A Stabilization Device-assisted Imaging Technique to Obtain Multi-dimensional Images of Blood Vessels in Mouse Liver

After confirming a lack of righting reflex in an anesthetized 8-week-old male C57BL/6 mouse, wipe the mouse's tail with 75% alcohol and use a 1-microliter syringe equipped with a 30-gauge needle to inject 10 milligrams per milliliter of freshly prepared Rhodamine B isothiocyanate-dextran in 100 microliters of Rhodamine B isothiocyanate-dextran into the caudal tail vein.

When all of the solution has been delivered, use a cotton swab to apply pressure to the puncture site and use gauze wet with sterile water to soak the abdominal fur. Use a razor to shave the abdomen, making strokes in the direction of the fur, and place the mouse onto a 37 degrees Celsius, alcohol-disinfected heating pad.

To fix the mouse liver with a body organ imaging frame, first, place a clean 5-millimeter diameter suction cup in a fixed position and wipe the heating pad and suction cup with 75% alcohol. Connect the suction cup to the vacuum pump hose and turn on the pump.

On a 75% alcohol-sterilized table, use surgical scissors to cut 2 centimeters of skin from the lower sternal border of the mouse and expose the liver. Place the mouse and the heating pad onto the holder base of the body frame and adjust the organ imaging fixture so that the suction cup can hold the liver. Then, use a negative pressure of 30 to 35 kilopascals for suction so that the liver attaches to the cup.

To set up the multiphoton laser scanning microscope, turn on the microscope and select the 60x objective. Fix the frame and the mouse under the objective and add a drop of normal saline to the cup large enough to cover the lens. Adjust the objective so the lens just touches the normal saline and turn on the laser software.