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 permeabil