Ocular Tissue Embedding and Cryosectioning in Optimal Cutting Temperature Compound for Fluorescence Imaging

After cryo protection of the rabbit eye is complete, transfer it from a 30%sucrose solution to a 100 millimeter dissection dish. Under a dissecting microscope, carefully flip the eye to remove excess sucrose solution from the inner part of the eye. Using a wipe, gently wipe away excess sucrose solution from the outer part of the eye, fill a disposable embedding mold with optimal cutting temperature or OCT compound to a depth of 0.5 to one centimeter.

Place the eye in the embedding mold facing upward and slowly fill the inner part of the eye with OCT compound, avoiding bubble formation. Once the eye is emerged in the OCT compound, and does not touch the inner surfaces of the mold, wrap the embedding mold in paraffin and store it at four degrees Celsius overnight. The next day, place the embedding mold in a dissection dish.

If possible, gently remove the OCT compound from the inside of the eye with a wipe. Transfer the eye to a new embedding mold filled with fresh OCT compound. Then fill the mold as previously done to ensure the final embedding medium is free of debris or imperfections.

Label the mold for orientation purposes. Position the eye upward in the cryo mold, so the OCT compound lines the inner retina. Use the optic nerve head as a landmark for determining the superior part of the eye Place the labeled embedding mold on dry ice, ensuring that the eye is not touching the inner surfaces of the mold or exposed to air.

Mount the block on the cryostat with the superior part of the eye facing up and the inferior part down. With the remaining cornea and iris facing to the right or left. Position a fresh blade parallel to the vertical axis of the eye to section it.

After cutting the sections and drying overnight, place a slide in a freezer at 80 degrees Celsius. The fluorescence images of a whole retinal cryosection of rabbit eye demonstrated a uniform pigmented retinal pigment epithelium layer without significant detachment from the overlying photoreceptor layer. A bright-field image of the retinal section further demonstrates intact retinal morphology of all layers.