To begin, place a formal and fixed mouse eye on a piece of wax paper under a dissecting microscope. With a pair of micro forceps, hold the remnants of the muscle and optic nerve. Then orient the eye so that the cornea faces one side.
Now use a surgical blade to make a one to two millimeter long incision behind and parallel to the limbus. Apply a downward force with the blade while holding the eye in place until the anterior segment separates from the posterior end. Then discard the anterior segment and lens.
Transfer the posterior segment into a six centimeter dish filled with PBS. Use a micros spatula to scoop out the retina while simultaneously holding the optic nerve with micro forceps. To rinse the retina, transfer it into a well of a 12-well plate that has six wells filled with double distilled water.
Use a P1000 pipette to carefully pipette water up and down adjacent to the retina while blowing the water to cause gentle agitation. Next, use an inverted glass pipette to transfer the retina to the second well. To digest the retina, transfer the rinsed retina into trypsin solution in a 12-well plate with an inverted glass pipette.
Center a trypsin-soaked P200 pipette tip on the optic nerve. Then gently pipette the entire vascular network up and down to dissociate the nonvascular tissue. Now, use an inverted glass pipette pre rinsed in trypsin to transfer the retinal vasculature into a well containing double distilled water in a six-well plate.
Swirl the plate, then pipette the vasculature up and down with an inverted trypsin-rinsed glass pipette to remove any residual nonvascular tissue. Carefully transfer the retinal vasculature to the center of the marked region on a charged surface microscopy slide. Then use a P200 pipette to carefully remove the excess water from one edge.
Place the slide in a biosafety cabinet close to the front side to let the residual water evaporate. When the vasculature is almost dry, transfer it under a phase contrast microscope. Use a P200 pipette to slowly add double distilled water on one side of the marked region to carefully rehydrate the vasculature.
Fixation of the isolated retinal vasculature resulted in structurally robust and sufficiently durable tissue suitable for AFM measurements. In contrast, the retinal vessels isolated from unfixed or briefly fixed eyes became fragmented and collapsed.
