Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

These methods can help answer key questions in the ophthalmology field such as pericyte proliferation or migration. The main advantage of these procedure is that they provide a variety of visualization options. Demonstrating the procedures will be Karin Dreisig and Frank W.Blixt, two post-docs at our laboratory.

To enucleate the eye, first use a scalpel to make the posterior and anterior slits of approximately 0.5 centimeters in the rat eyelid. Grab the eye with forceps and tilt carefully to the side to expose the surrounding tissue. Enucleate the eye by making cuts with dissection scissors in the connective and muscular tissue.

Briefly place the eye in 4%formaldehyde and phosphate-buffered saline for one to two minutes. Rinse briefly in sodium buffer before making a hole at the corneal limbus by applying light pressure with the tip of a scalpel. Cut along the corneal limbus with dissection scissors to remove the cornea and lens with forceps.

Submerge the remaining tissue with retina in 4%formaldehyde in PBS for two to four hours. Cryo-protect the tissue by immersing in Sorenson's phosphate buffer containing 10%sucrose and then 25%sucrose. Transfer the tissue once it has sunk to the bottom of the container.

Embed in Yazulla medium prepared with 3%gelatine from porcine skin and 30%albumin from chicken egg white. Next, cut 10-micron vertical cryosections of the gelatin-embedded retina through to the optic nerve. Place the cryosections on a glass slide and allow to dry for a minimum of one hour.

Once dry, submerge the slide in PBS with 0.25%Triton-X 100 for 15 minutes. Then, drip a mixture of 1:100 PDGF receptor beta and 1:500 NG2 primary antibodies in PBST and 1%BSA onto the cryosection and place in a moist chamber at four degrees Celsius overnight. The next day, wash the sections by immersing the slide in PBST twice for 15 minutes each time.

Then drip a mixture of 1:100 Anti-Mouse Alexa Fluor 594-linked and 1:100 Anti-Rabbit FITC-linked secondary antibodies diluted in PBST with 3%BSA onto the cryosections and incubate for one hour in the dark. After the incubation, rinse the glass slide in PBST twice for 15 minutes each time. Mount the stained cryosections with anti-fading mounting medium containing DAPI and a coverslip.

A second retinal tissue preparational technique is whole-mount. Following the removal of the cornea, use small opening movements of forceps to separate the retina from the retinal pigment epithelium towards the optic nerve. Free the retina at the optic nerve with dissection scissors and make three to four slits of a few millimeters'length from the retinal periphery towards the optic nerve head.

Spread the retina onto a glass slide and allow to dry for five to 10 minutes. Drip 4%formaldehyde onto the retina and fix for 20 to 30 minutes. Following fixation, rinse with PBS.

For optimal results, immunostain directly after rinsing. First, drip PBST onto the whole-mount and incubate at room temperature for 15 minutes. Pour off the PBST, then add the primary antibody solution and incubate in a moist chamber at four degrees Celsius overnight.

The next day, pour off the primary antibody and drip on PBST to rinse the retina twice for 15 minutes each time. After pouring off the second wash, drip secondary antibody solution onto the retina and incubate for one hour in a moist chamber at room temperature in the dark. Following the secondary antibody incubation, wash twice with PBST as before.

Mount the stained whole-mount with anti-fading mounting medium containing DAPI and a cover slip. Alternative to whole-mounting the retina, the retinal vasculature can be isolated by hypotonic isolation. First, place the retina in one milliliter of deionized water in a 24-well plate and shake at 200 rpm with a 1.5-millimeter vibration orbit for one hour at room temperature, then add 200 units of DNAse 1 to dissociate the lysed cell debris from the retinal vasculature and shake for another 30 minutes at room temperature.

Rinse the retina in deionized water for three times for five minutes each with shaking at 150 to 300 rpm to remove neuronal cell debris. The retina should become more transparent with each rinse, indicative of the removal of neuronal cellular debris. After rinsing, fix the retina in one milliliter of 4%paraformaldehyde and PBS for 10 minutes at room temperature.

Rinse with PBS three times. If proceeding to immunostaining, block the hypotonic-isolated vasculature with 500 microliters of 10%donkey serum diluted in PBS for one hour with shaking at 100 rpm. After blocking, incubate the retina overnight in 600 microliters of primary antibody solution consisting of 1:100 PDGF-receptor beta and 1:500 NG2 primary antibodies diluted in 10%donkey serum and PBS.

The next day, rinse the retinal network three times in PBS for five minutes each time, then incubate in 1:100 Anti-Mouse Alexa Fluor 594th-linked and 1:100 Anti-Rabbit FITC-linked secondary antibodies, diluted in 10%donkey serum and PBS with shaking at 100 rpm at room temperature for one hour in the dark. Following a five-minute wash in PBST, incubate in 0.2 nanograms-per-milliliter DAPI and PBST for 15 minutes. Wash three times for five minutes with PBST while protected from light.

Next, cut the tip of a plastic Pasteur pipette. Remove sharp edges using a pipette tip, and moisten the Pasteur pipette with PBST, then aspirate the retinal vasculature into the pipette and dispense into a four-well glass chamber slide. The moistening step is important to avoid the retinal vasculature sticking to the inside of the Pasteur pipette.

Position the retinal vasculature by tilting the chamber slide back and forth. Remove the medium from the wells of the chamber slide. The surface tension of the liquid will flatten the vasculature onto the bottom of the slide.

If needed, drip drops of liquid onto the retinal vasculature to unfold. Ensure correct unfolding under a microscope before removing the plastic wells from the chamber slide. Mount the stained vasculature with anti-fading mounting medium and a cover slip.

NG2 immunohistochemistry reveals positive vessels within the inner part of the retina. Arrows indicate circular and horseshoe-shaped immunoreactivity in the vessels. The arrowhead points out a longitudinally-cut vessel.

PDGF-receptor beta immunohistochemistry shows similar immunoreactivity to NG2. Again the arrows and arrowhead indicate immunoreactivity in the vessels. This image shows a merge of NG2 in green, PDGF-receptor beta in red, and DAPI in blue.

By superimposing the three different filters, it is revealed that NG2 and PDGF-receptor beta are co-localized. This image shows an immunostained whole-mount preparation with NG2 staining in red. Immunoreactivity is visible along the superficial vascular network with intense staining in abluminal pericytes.

The neuronal cells are visible as DAPI blue nuclei between the NG2-stained microvasculature. This image shows the hypotonic isolated rat retinal vascular network stained with NG2 in green and PDGF-receptor beta in red. The complete network showed NG2-immunoreactivity.

PDGF-receptor beta immunoreactivity was found in cell somas. At higher magnification, it is clear that PDGF-receptor beta staining is only seen in cell somas in the vascular network, indicating pericyte immunoreactivity. Including DAPI staining together with NG2 and PDGF-receptor beta reveals three pericytes in two undefined vessel wall cells in this high-magnification image.

Following these procedures, other immunostainings like smooth muscle cell or endothelial stainings can be used in order to answer additional questions. For example, how do cells in the retinal vasculature look following ischemia?