Preparation of Mouse Retinal Cryosections to Assess Retinal Permeability to Adeno-Associated Virus

Take an eye harvested from a mouse model of a disrupted retinal barrier, preinjected with adeno-associated virus carrying the green fluorescent protein gene.

The disrupted retinal barrier allows AAV to penetrate photoreceptor cells, where the viral genome is expressed to produce GFP, making the cells fluorescent.

Dissect the eye to remove the cornea and lens, leaving the eyecup intact.

Immerse the eyecup in a fixative solution to preserve retinal structure.

Transfer the eyecup sequentially through increasing concentrations of cryoprotectant solutions. Later, incubate in the final concentration to prevent ice crystal formation.

Embed the eyecup in cryo-medium and freeze it.

Using a cryostat, section the eyecup into thin slices.

Mount the section onto a slide. Add a buffer containing detergents to permeabilize the tissue to allow antibody access during subsequent immunostaining.

Rinse the section. Treat it with a blocking solution to prevent nonspecific antibody interactions during the immunostaining.

The retinal cryosection is ready for further immunostaining procedures and fluorescence imaging. GFP-positive fluorescent cells reveal successful viral transduction, indicating retinal permeability.

After harvesting the eyes from a euthanized injected animal, dissect the eyes to remove the lens and cornea. Then immersion-fix the dissected eye in 4% paraformaldehyde for an hour. After an hour, cryo protect the eyes in 10% sucrose for another hour at room temperature. Then soak the eyes in 20% sucrose for one third of an hour at room temperature.

Finally, leave the eyes in 30% sucrose solution overnight at four degrees Celsius. The next day, embed and freeze the eyecups in an embedding resin such as cryo matrix. Then using the cryostat, make 10 micron sections and mount them to slides prepared for frozen tissue.

Now permeabilize the sections on the slides with detergent for five minutes. Follow this with two brief washes in PBS. Then block the eyes for an hour at room temperature.