A Technique for the Prolonged Incubation of Neuronal Tissue of the Retina

JoVE Encyclopedia of Experiments

Neurowetenschap

0 weergaven • 3:30 min • July 8th, 2025

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Take an intact mouse eye in artificial cerebrospinal fluid, or aCSF, and incise to isolate the eyecup containing the retina.

Treat with an enzymatic solution to remove the inner limiting membrane, exposing the neuronal cells.

Immerse the tissue in an inhibition solution to stop digestion, and wash to remove excess solution.

Place the tissue in a specialized incubator at a low temperature to slow cellular metabolism, preserving the tissue.

This incubator comprises a main chamber with a mesh for mounting tissues. The chamber is filled with aCSF, and its temperature and pH are controlled to preserve tissue functionality.

To eliminate microbial contamination, the aCSF is pumped into another chamber and treated with ultraviolet radiation to kill any microbes.

The sterilized aCSF is then pumped back into the main chamber.

To isolate the retina, remove the eyecup, separate the retina from the surrounding tissue, and return it to the incubator.

For retinal whole mount preparation, immediately enucleate the eyes from a euthanized animal. Make a small cut along the ora serrata, and place the eyes in either Ames Media or aCSF with carbogen supply at room temperature. After that, immediately remove the cornea, lens, and vitreous by cutting along the ora serrata with small scissors, and remove them with forceps. Place the tissue in the incubation system at room temperature.

To remove the inner limiting membrane, transfer the eye cup containing the retina to a small glass jar containing papain, L-cysteine, EDTA, and DNAse at 37 degrees Celsius for 20 minutes. Apply carbogen to the solution through the lid, but do not bubble. Stop the enzymatic digestion by placing the tissue in an ovomucoid and BSA solution for 10 minutes in Earl's BSS. Then, wash the tissue thoroughly with aCSF.

Subsequently, transfer the tissue to the incubation system and reduce the temperature to about 15 to 16 degrees Celsius. Following that, transfer the retinal tissue to the microscope; isolate the retina from the eye cup, and cut it into four pieces with a razor blade. If the entire retina is required, make four small cuts in the periphery of the retina to allow it to lie flat.

This tissue can now be transferred to the microscope for electrophysiology, or maintained in the Braincubator. The tissue is maintained in the main chamber containing the probes for pH and temperature measurements. A second chamber is isolated from the main chamber and is exposed to 1.1 watts UVC light in order to irradiate bacteria floating in the solution. Use a peristaltic pump to circulate aCSF through the two chambers, and a Peltier thermoelectric cold plate to either cool or heat the main chamber.

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Laatst bijgewerkt: 15 augustus 2026