1. Retinal Preparation

1. Enucleate the mouse eye. Using one hand, open the eye lids so that the eye is visible. With the other hand, using a curved forceps (curved facing upwards), apply pressure on superior and inferior aspects of the orbit until the globe protrudes. Gently close forceps at the posterior aspect of the eye and lift in a continuous motion to enucleate the eye.
2. Fix eye with 10% neutral buffered formalin for at least 24 hr.
3. Place eye in PBS solution in a small Petri dish.
4. Dissect out retina carefully under a microscope, taking care to avoid inducing large tears. Make an initial cut in the cornea with dissection scissors. Then, while holding the cut area with straight forceps, use scissors to cut along the border of the cornea and sclera removing the cornea. Using fine curved and straight forceps, carefully peel the sclera and choroid away from the retina towards the optic nerve. This step requires patience as quickly freeing the retina can lead to tears. Remove the lens and any excess iris and debris from the retina ¹⁶.

2. Water Washes

1. Place retina in a 24-well dish and cover with water (approximately 500 μl, filtered with at least a 45 μm filter).
2. Change water every 30 min-1 hr, at least 4-5 times.
3. Leave overnight in water with gentle shaking at room temperature.

Note: The washing steps are very important as they help separate the neural retinal layers from the blood vessels.

3. Trypsin Digest

1. Remove water and incubate retina in a solution of 3% trypsin (Difco 1:250) in 0.1 M Tris buffer (pH 7.8) at 37 °C with gentle to no shaking. Trypsin digest is completed when the tissue begins showing signs of disintegration (approximately 1.5 hr).
   Note: Avoid rapid shaking as this can damage the vasculature.
   
2. Carefully, pipette out trypsin into a separate well using a microscope to avoid touching the retina. This excess trypsin can be used later, when manipulating the vessels under the microscope, in step 4.3.

4. Separating the Vasculature

1. Add filtered water to the retina. Then, attempt to peel off the internal limiting membrane with forceps, using scissors if necessary. Shake with mild agitation for 5 min. Carefully, pipette out the water under the microscope to avoid damaging the retina.
2. Repeat water washes (for 5 min each) to help free the network of vessels from the adherent retinal tissue. Water washes are completed when there is little to no debris remaining in the water after wash.
3. Careful manipulations under a dissection microscope are now needed to further free up the network of vessels. Below are several useful techniques, which may be used in sequence or individually to achieve the desired outcome based on technical preference:
   1. Using a 200 μl pipette, carefully pipette water up and down adjacent to vessels, blowing water at vessels, causing gentle agitation.
   2. Using 200 μl pipette, pipette the trypsin up and down to help coat walls of the pipette. Then, CAREFULLY pipette the entire vessel network up and down once to help break down non-vascular tissue.
      Note: Centering the pipette tip at the optic nerve rather than the edges of the vascular network can also prevent the vasculature from sticking to the tip.
      
   3. Gently lift vasculature with fine forceps out of water to dislodge non-vascular components.
   4. If the above steps do not work, add trypsin to the retina to help break down the tissue, and incubate at 37 °C for several minutes. Then, remove trypsin and repeat steps 4.1-4.3.

Note: Make sure to coat all instruments that will come into contact with the vessels in trypsin (e.g. pipette tip, forceps). This will help avoid adhesion of vasculature to the equipment.

5. Visualizing the Vasculature

1. Place a drop of water onto a clean slide. Using a 200 μl pipette or forceps, transfer the digested vessels into the water. Make sure the vascular flat-mount is unfolded. The surface tension of the water helps to unfold the now diaphanous vascular network. If the tissue does not unfold, add additional water and gently shake the slide to facilitate unfolding.
2. Remove excess water very slowly by pipetting or using a paper towel.
3. Allow slides to dry completely.
4. Stain slides via appropriate protocol with either PAS/hematoxylin or H&E stain.
   Note: PAS/hematoxylin is useful for identifying vessel wall and cellular nuclei; H&E is useful for demonstration of RBC as well.
   
5. Dehydrate and mount (Permount mounting medium).