The present protocol describes an in vivo paracellular permeability detection measure to evaluate the function of tight endothelial junctions in mouse submandibular glands. Two photon laser scanning microscopy, nevertheless, not only has the advantage of conventional confocal microscopy but can also be used to detect deeper tissue and image more clearly. This experiment provides a good method measure for assessing the vascular permeability of different tissues especially the surface tissues and organs of animals.
Begin by choosing appropriate tracers such as fluorescein, isothiocyanate, labeled dextran and Rhodamine B, labeled dextran with distinct excitation emissions spectra, to minimize the interference between fluorescence signals for the permeability assay. Dilute the traces into sterile phosphate buffered saline into 100 milligrams per milliliter stocks and store the aliquots protected from light at minus 20 degrees Celsius. After anesthetizing the 8 to 10 weeks old male wild type mouse, gently hold the head and neck of the mouse to make one side of the eyeball protrude slightly.
Insert an insulin syringe containing 100 microliters of fluorescent tracer solution mixture along the corner of the eye at a right angle to the eye. After the injection, quickly place the mouse on the cardboard in a supine position with its limbs and head taped. For submandibular gland or SMG isolation, under a stereoscopic microscope, cut the epidermis of the neck with general tissue scissors to expose both sides of the SMGs.
Next, use a blunt tissue separation needle to gently separate the capsule from the gland surface without damaging the gland tissue, blood vessels, and glandular structure. Connect the customized holder to the negative pressure device and check whether the negative pressure effect is properly achieved in advance. Gently place the exposed SMG on the customized holder and suck up the tissue by negative pressure suction, as far away from the mouse body as possible, to reduce motion artifacts caused by respiration and other conditions.
Commence imaging soon after the site of the gland is chosen. Acquire sequential images along the Z axis stepped two micron apart from the gland's surface up to 70 to 140 microns into the tissue to generate a three-dimensional construction. Next, acquire time lapse imaging of the vessels to measure vascular permeability in the SMG.
In the Explorer dialogue box, click on Add New Folder"and change the file name. Then, go back to the acquisition column. In XY, the format is 512 by 512, and the speed is 400 hertz.
Turn Bidirectional X"on, set the zoom factor to 0.75 and 1.5 for zoom. Next, select XYT under Acquisition Mode"to acquire time lapse imaging. Set duration to 20 seconds, 30 minutes or longer according to the experimental need.
After setting the conditions, click on Live"to observe the vascular imaging of two channels and overlapping channels in the photo forming frame and choose the areas of interest. Click on Start"to begin imaging. In vivo vascular permeability assay, and three dimensional images of blood vessels in mice.
Submandibular glands are shown here. In the control group, both the tracers existed in the blood vessels of the SMG. Due to its small molecular weight, FD4 was able to leak out of the blood vessels to the basal sides of acini and ducts, thereby clearly depicting the shape of the acini and ducts.
The RD70 was distributed in large sized blood vessels and micro vessels. In the duct ligation group, both FD4 and RD70 were extravasated to the basal sides of acini, which indicated that duct ligation could disrupt the endothelial barrier function and increase the permeability to large molecules. Further, the semi-quantitative FD4 and RD70 fluorescence intensity results confirmed these observations.
The three dimensional images showed much more obscured fluorescence of FD4 and RD70 around blood vessels in the ligation group compared to the control group. Careful SMG isolation and appropriate placement and the microscope are essential prerequisites for successful detection. Following the procedures, the blood flow rate can be calculated by marrying the movement of red blood cells and the infiltration of the fluorescence labeled blood cells can be chased.
