Two to three weeks before imaging, the vitreous of an anesthetized rat's eye is injected with the viral particles carrying the genetically encoded calcium indicator. Using fundoscopy and OCT, the retinal structure is examined for adverse reactions from the calcium indicator injection. Two weeks after the injection, the ganglion cells layer or GCL exhibited fluorescence emission.
Then the retina excision from the euthanized rat is initiated. Take the eyes expressing AAV2-CAG-GCaMP5G and remove all tissue surrounding the eyeball using small curved forceps and fine spring scissors. Next, take a three by three centimeter piece of filter paper.
Position it on the cover of a 3.5 centimeter dish and drench the filter paper with AIMS medium. Position the eyeball on the paper with the anterior segment oriented towards the operator. Use a pair of straight forceps to hold the eyeball at approximately a 45 degree angle from the dish surface.
Use the gap between the straight forceps to guide an incision with a blade. Then immerse the eyeball into AIMS medium. Use straight forceps and fine spring scissors to separate the anterior and posterior segments of the eye.
Remove the lens carefully using two pairs of straight forceps and detach the retina from the sclera. Using fine spring scissors, make an incision in the sclera towards the optic nerve and cut until the retina has been successfully separated from the eye cup. Transfer the excised piece of the retina onto the mounting membrane using a cut tip plastic pipette.
Then use a pair of straight forceps to flat mount the retina, ensuring the ganglion cell layer is oriented upwards. Next, use a plastic pipette fitted with a 100 microliter pipette tip to remove the media, facilitating adherence of the retina piece to the porous membrane. Next, flip the assembly onto the micro electrode array or MEA, positioning the GCL on top of the electrodes.
Then fill the sample bath with oxygenated AIMS medium.
