To begin, load five microliters of hoechst solution into a sterile 10 microliter glass Hamilton syringe, attached to a 34 gauge blunt needle. Insert this syringe needle into the anterior chamber through the self-sealing corneal tunnel, and inject the solution. Hold the needle in place for two to three seconds, until all fluid clears.
Pull out the needle slowly to remove it, avoiding leakage. Next, enucleate both eyes. To isolate the corneas, cut the sclera around the optic nerve, and make four additional cuts from the optic nerve towards the cornea.
Drain the excess fluid with the Whatman filter paper. Spread out the cut sclera, and separate the retina from the sclera. Pull out and discard the lens and retina.
Next, remove the iris. Cut and discard the sclera around the cornea. And carefully lift up the cornea.
Placing the corneas on a glass slide, stain them with 0.5%Alizarin Red to identify endothelial cells. After removing the excess stain, wash the cornea three times, and place a cover slip on it. Examine the corneas under a light microscope to image the Alizarin Red staining of endothelial cells.
Also, evaluate the corneas under a fluorescent microscope to observe hoechst staining, and compare it to the non-injected cornea as control. Hoechst dye, a DNA binding cell permeable fluorescent marker, was utilized to assess drug bioavailability through intracameral injection. Corneal endothelial cell uptake of hoechst was examined 15 minutes post-injection by fluorescent microscopy.
Alizarin Red staining highlighted the intercellular borders of the endothelial layer. Also, positive hoechst staining within corneal endothelial cell nuclei confirmed the successful delivery of the dye via this method.
