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

In This Article

Abstract

Source: Vacca, O., et al. Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease. J. Vis. Exp. (2015)

This video demonstrates the step-by-step procedure for preparing mouse retinal cryosections to visualize adeno-associated virus permeability across disrupted retinal barriers.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

For retinal cryosections (Figure 1), dissect enucleated eyes to remove the lens and cornea, and immersion fix in 4% paraformaldehyde for 1 hr.
1. Cryoprotect the previously fixed eyes in 10% sucrose for 1 hr at room temperature, 20% sucrose for another hr at room temperature. Cryoprotect in 30% sucrose solution, overnight at 4 °C. Freeze and embed eyecups in embedding resin such as Cryo-matrix. Make 10 µm cryosections and mount on slides specially treated for frozen tissue sections and which improve tissue adherence during staining.
2. Permeabilize sections with 0.1% Triton X-100 in phosphate-buffered saline (PBS) pH 7.4 for 5 min. Wash 2x in PBS and block for 1 hr at room temperature in PBS with 1% bovine serum albumin (BSA), 0.1% Tween 20.

Results

Figure 1: Schematic representation of the general protocol. After adeno-associated virus (AAV) production, particles are injected into the vitreous or into the penile vein to anesthetize adult mice. Fundus images with a Micron III camera are performed to follow the green fluorescent protein (GFP) expression. The GFP expression pattern is analyzed on retinal cryosections and flatmounted retinas thanks to immunohistochemistry and confocal images. The AAV passage through the blood-retinal barrier (BRB) is assessed by polymerase chain reaction (PCR) analysis on blood samples coming from intravitreally or intraperitoneally injected mice, in order to detect the presence of GFP sequence, indicator of the AAV particles presence into the bloodstream.

Materials

List of materials used in this article
Name	Company	Catalog Number	Comments
Cryomatrix embedding resin	Thermo Scientific	6769006
Superfrost plus adhesion slides	Thermo Scientific	10143352
Binocular magnifier SZ76	ADVILAB	ADV-76B2	Zoom 0.66 x 5 x LEDs with stand epi and dia / Retinas dissection
Spring scissors straight - 8.5 cm	Bionic France S.a.r.l	15003-08	Retinal dissection