Intravital Fluorescence Microscopy to Study Microvascular Thrombus Formation

Place the animal on the platform under the stereomicroscope. Use 10X magnification. For microscopy of the right ear, prepare the left jugular vein by first creating a 5-millimeter incision in the skin of the neck in a craniocaudal direction. Then, use micro-forceps and microscissors to dissect the subcutaneous tissue. Then, free the vein from its adventitia without touching the vessel.

Now, use the previously prepared insulin syringe for the injection of the fluorescent dye. Carefully grab the vessel wall with the micro-forceps without perforating the vein. Penetrate the distended vessel wall with the syringe needle, and inject FITC-dextran intravenously. Withdraw the needle, and stop the bleeding using cotton swabs. Avoid blood and dye contamination of the ear.

Transfer the animal on the heating plate to an acral glass construction with a slot for the heating plate and a 0.5-centimeter high plane for positioning the ear. Fix the animal in a prone position on the heating plate using adhesive bandage. Place the convex cartilage at the base of the ear beside the plane for the ear so that the apical part of the ear can be positioned flat on the plane. Add one drop of room temperature aqua to the acral glass plate. Using cotton swabs, absorb the drop of aqua and let capillary forces attach the ear plane to the acral glass.

The auricle must be positioned as flat as possible, even though the cartilage gives the auricle a convex shape. So, only the more flexible distal part of the auricle should be positioned on the acral glass plate. To avoid tissue damage and extravasation of the frozen dye, touch the ear as little as possible with the forceps.

Next, add one drop of aqua to the convex dorsal side of the ear. Carefully put one coverslip on the ear without compressing the basal vessels entering the ear. Use cotton swabs to remove as much aqua as possible from under the coverslip, to minimize the distance between the coverslip and the ear target vessels.

Begin by adjusting the intravital fluorescence microscope for FITC-dextran visualization. Transfer the prepared animal to the desk of the intravital fluorescence microscope. Using 5X, 10X, and 20X magnification and 20% light intensity, search for a venous vessel of 50 to 60 microns in diameter and with an anterograde blood flow.

Add one drop of room temperature water to the coverslip for water immersion of the 63X magnification objective. Immediately after the application of the water drop, begin recording the vessel for 20 seconds for the baseline assessment of the diameter and blood flow. Start thrombus induction 5 minutes after the injection of FITC-dextran. For this purpose, raise the light intensity to 100%.

During phototoxic thrombus induction, close the aperture of the microscope for 2 seconds within a 30-second period to check for occlusion of blood flow. If blood flow persists, open the aperture again. The vessel is classified as occluded if the flow stands still for 30 seconds or more, or if the blood flow is retrograde. Select and occlude five vessels per ear within a 1-hour period after injection of FITC-dextran.