The overall goal of this procedure is to investigate angiogenesis in the neonatal mouse retina. This is accomplished by first nucleating postnatal eyes, and then applying a fixative of the third step is to carefully dissect the postnatal murn retina into a petal shape, and the final step is to stain the retinal vasculature. Ultimately, visualization of mouse retina vasculature is achieved through fluorescent staining and confocal microscopy.
Demonstrating this procedure will be Dr.Simon Tlo, a post-doc in my laboratory, and Kathleen Allenson, a previous member of my laboratory. Throughout this procedure, the default temperature is room temperature. Begin by placing a humanely killed mouse pup on its side with scissors.
Remove the skin covering the eye. Next, with the scissors positioned below the eye, cut the optic nerve and surrounding tissues and lift out the eye. Transfer the enucleated eye to a 24 well plate containing fixative, which is 4%PFA in two times.
PBS proceed with dissecting the next eye while one is in the fixative. The stagger between the two eye dissections will be half a minute. With practice.
Allow each eye to fix for 10 to 15 minutes. Then transfer them to cold two times PBS on ice for at least five to 10 minutes. Before isolating the retinas, collect as many eyes into the PBS as desired, using a plastic pasture pipette that has been cut to widen the boar.
Transfer the eyes into a Petri dish containing two times PBS under a dissecting microscope. Remove any fat around the eye using forceps and scissors. Pierce the edge of the cornea with sharp scissors and cut around the cornea and iris, discard these tissues.
Next, insert the forceps between retina and sclera. Remove the sclera while taking care not to tear the retina. The choroid layer can also be removed as long as there is no risk of damaging the retina.
Leaving the choroid layer on should not significantly affect the quality of the immuno staining. Now using forceps, remove the lens and the vitreous humor. Follow with removing the aloid vessels.
Use fine forceps to gather them together in the center of the eye. Then quickly pull them away from the center of the retina. Take extra care to remove all the yellow thistle because if you leave some behind, they can obscure your standing results.
The cup shaped retina should now be transferred to a clean Petri dish and rinsed clean with PBS. Now into the retina. Make four to five radial incisions that reach about two thirds of the way towards the center.
Use spring scissors to create this pedal shaped retina. This step takes patience and practice to perfect. Now draw off most of the PBS using a pasture pipette.
This will flatten the retina. Then remove any excess PBS with a small piece of absorbent paper. Next, apply minus 20 degrees methanol drop by drop onto the surface of the retina until it is mostly submerged, and then liberally add methanol.
The retinas will turn white. This step helps to fix the retinas and will facilitate permeation. Transfer the retinas in the cold methanol to a small tube.
Using a wide bore plastic pipette. Allow them to incubate in the cold methanol for at least 20 minutes if needed, the retinas can stay in the methanol at minus 20 degrees for several months. The antibody use here only.
The cine cannot be used successfully on maternal fixed retina. In this case, retina needs to proceed straight to immunostaining after flattening them to a petal shape. Begin by carefully pipetting off the methanol from the retinas.
Then give the retinas a quick and gentle rinse in one times PBS. Remove the PBS and cover the retinas with 100 microliters of perm block solution with 5%of the appropriate serum. Set the retinas to shake gently for an hour later.
Remove the perm block solution with a pipette and replace it with 100 microliters of perm block solution containing the primary antibody or is selectin before. Then set the retinas to incubate overnight at four degrees Celsius with rocking the next morning. Wash the retinas four times in PBS TX for 10 minutes per wash.
Apply gentle rocking during all the wash steps. If the primary antibody is unconjugated, add 100 microliters of the appropriate fluorescent secondary antibody, diluted one to 200 in PBS tx. Incubate the retinas overnight at four degrees Celsius or for four hours at room temperature with gentle agitation to remove the secondary antibody.
Wash the retinas four times in PBS TX with gentle rocking for 15 minutes per wash. Now proceed with mounting. Transfer the retinas onto slides using a wide bore plastic pipette and remove any excess P-B-S-T-X with a tissue.
Then mount the retinas in 50 microliters of prolonged gold and gently cover the solution with a cover slip. To allow the prolonged gold de set refrigerate the slides overnight at four degrees Celsius, protected from light on the following day. Proceed with imaging the retinas after dissection.
The retina should look like a flat flower using ISO selectin B four Alexa 4 88. The endothelial cells were stained and appear green in color. Supporting vascular muscle cells were visualized using Antifa smooth muscle, actin the low power view using a five times objective, allowed an overview of the vascular organization of the retina by using a different combination of antibodies.
Endothelial cells can be seen using anti CD 31 immuno staining, supporting parasites where visualized using an antibody against NG two. Alternatively, the supporting parasites were stained with anti desmin. Anti desmin staining visualizes the finger-like processes of the parasites and helps determine whether they're closely associated with the endothelial cells.
In contrast, anti NG two staining outlines the nuclei of each parasite, which is useful when quantifying the number of parasite cells in the vasculature. While attempting this procedure, it's important to remember to avoid touching and damaging the retina surface with your dissection tools. Following this procedure, other method like BD staining can be performed in order to investigate proliferation of specific cells.
