Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO⁺ and Light Sheet Fluorescence Microscopy

Our protocol provides three-dimensional and large-scale views of vertebral lymphatic vessels and their draining lymph nodes, facilitating the study of lymphatic drainage and the lymphatic traffic of immune cells. The iDISCO+clearing protocol preserve tissue integrity, allowing localization of the neural, vascular and immune cells of the vertebral column, including the spinal cord, meninges, epidural space and draining lymph nodes. After confirming a lack of response to pedal reflex in an anesthetized 8 to 12 week old mouse, place the mouse in a stereotaxic frame.

And use a scalpel to make a skin incision in the appropriate region for the planned injection. Use a spinal adapter to immobilize the spinal cord at the Th12-L1 vertebral level and blunt dissect the paracervical or paraspinal muscles covering the neck and the column to visualize the surface of the dura mater. Then use a 26 gauge needle to carefully punctuate the central area of the dura mater and under-laying arachnoid.

Next, cut two millimeters from a glass capillary tip and connect the capillary to a cannula linked to a 10 microliter syringe. Load the micro capillary with two to eight microliters of the fluorescent tracer of choice. And introduce a micro capillary into the medial region of the dura mater at the appropriate angle for the planned injection.

Pushing the capillary 1.5 millimeters below the dura mater, close the incision around the capillary with 10 microliters of surgical glue. When the glue has dried slowly inject the fluorescent tracer at a rate of one microliter per minute. When the entire volume has been delivered leave the capillary in place for one minute before retracting the micro-capillary.

And close the injection hole with additional glue. At the appropriate time point after the injection use dissection scissors to make skin and peritoneal incisions from the lower abdomen to the thoracic cage. Open the thoracic cage to access the heart and insert a 26 gauge needle into the left ventricle of the heart.

Perfuse with 20 milliliters of ice cold 4%paraformaldehyde in PBS, at a rate of two milliliters per minute. And use scissors to rapidly cut the right atrium releasing the perfusion fluid stream. Use forceps to completely remove the skin and remove the limbs with scissors.

Remove all of the internal organs, taking care to leave the lymph nodes intact and cut the ribs. Immerse the dissected tissues in ice cold 4%paraformaldehyde in PBS in individual 50 milliliter tubes overnight at four degrees Celsius, before washing the fixed tissues three times in 50 milliliters of fresh PBS, for five minutes per wash. For iDISCO+labeling of whole-mount samples first, dehydrate the tissues with successive immersions in ascending concentrations of methanol in PBS for one hour per immersion with agitation at room temperature.

After the last immersion incubate the samples in a 33%methanol, 66%dichloromethane solution overnight with agitation. The next morning, wash the samples two times with 100%methanol for one hour per wash at room temperature. Followed by an overnight incubation in 5%hydrogen peroxide in methanol at four degrees Celsius.

The next morning rehydrate the samples gradually in sequential immersions in descending concentrations of methanol in PBS for one hour per concentration with agitation at room temperature. To decalcify the vertebrae incubate the samples in Morse's solution for 30 minutes at room temperature. Followed by two rinses in PBS and two, one-hour incubations, in 0.2%Triton X-100 in PBS, with agitation at room temperature.

After the second Triton X-100 incubation, treat the samples with permeabilization solution for 24 hours at 37 degrees Celsius. Followed by a 24 hour incubation in blocking solution at 37 degrees Celsius. At the end of the blocking solution incubation label the samples with primary antibody, diluted in a 0.2%Tween-20 and 0.1%heparin in PBS supplemented with 5%dimethyl sulfoxide and 3%donkey serum solution at 37 degrees Celsius for six days.

At the end of the incubation, wash the samples with four to five overnight incubations in fresh 2%Tween-20 and 0.1%heparin in PBS per wash at room temperature with agitation. Then incubate the samples with secondary antibodies according to manuscript directions. After the last wash dehydrate samples with successive immersions in ascending concentrations of methanol in PBS for one hour per concentration.

Followed by an overnight incubation in 33%methanol 66%dichloromethane solution. The next morning, wash the samples with two 15-minute dichloromethane washes. And clear the samples in dibenzyl ether without shaking for four hours.

For light sheet fluorescence microscopy imaging place the cleared samples in a transversal plane in the microscope stage under a 4X, 0.3 objective. And select a single sided three sheet elimination configuration with a fixed X position without dynamic focusing. Use LED lasers tuned to 561 nanometers and 100 milliwatts and 639 nanometers and 70 milliwatts.

And set the light sheet numerical aperture to 0.03. Set the appropriate emission filters and fill the microscope chamber with dibenzyl ether. Next, use the camera to acquire stacks with 2.5 micrometer Z stacks and a 30 millisecond exposure time per step using the two X optical zoom and 0.8 micrometers per pixel.

And perform the mosaic acquisitions with a 10%overlap on the full frame. Acquire images in TIF format and convert them into 3D format with an appropriate full conversion software program. To reconstruct the mosaics acquisition with Stitcher software open the images and manually arrange the images to reconstitute the whole mosaic picture, using the 10%overlap between the images as a guide.

Then use 3D software to generate orthogonal projections of the data. Adding a color-code attribute to the lymphatic vessels and the other anatomical structures on display. And set a gamma correction of 1.47 to the raw data obtained from the light sheet fluorescence microscopy.

according to the manufacturer's instructions. The combination of iDISCO+with light sheet fluorescence microscopy preserves the vertebral anatomy and allows capture of the lymphatic vasculature within the surrounding bones, ligaments, muscles and nerve ganglia. The injection of a small red fluorescent tracer into either the cerebral spinal fluid, the cisterna magna or the thoracolumbar region of the spinal cord results in tracer accumulation within the deep cervical lymph nodes 45 minutes after injection, indicating the uptake and drainage of the tracer by the lymphatic system.

Tracer injection into the thoracolumbar spinal parenchyma results in tracer accumulation within spinal cord tissues and deep cervical draining lymph nodes, but not in the cervical and thoracic lymphatic vasculature labeled with anti-LYVE-1 antibodies. Anti-LYVE injection into the thoracolumbar spinal parenchyma results in labeling of both the vertebral lymphatics and their extra-vertebral lymphatic connections. Substantiating the tracer uptake by vertebral lymphatic vessels and the lymphatic drainage towards the extra-vertebral lymphatic system.

Perform the trace injection very carefully to not to disturb that CSF circulation. The iDISCO+protocol is very officious but you need to adapt to the size of your samples. After trace injection into the central nervous system immune staining or live imaging experiment can be AUS-performed.

The iDISCO+protocol can be used to study different cells, populations, and tissues.