Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

This protocol allows for the investigation of the fine morphology of retinal neurons and can give insight into the cellular and subcellular morphological changes that occur in disease states. This method significantly improves the optical transparency of the retina and allows for high-resolution, three-dimensional imaging, circuit wiring, and fine sub cellular structures of retinal neurons in hormone retina preparation. Enucleate the mouse eyes with curved forceps and transfer them into a small Petri dish with 0.1 M PBS.

Poke a small hole along the cornea sclera junction with the needle under the dissection microscope, then transfer the eye into 4%power formaldehyde for one hour. Transfer the eye back to a dish with PBS. Under a dissection microscope, use dissection scissors to cut all the way around the Corneoscleral junction.

Remove the cornea and lens. Then cut it at the base of the optic nerve and carefully peel the sclera off with forceps to isolate the retina. Make four small cuts evenly around the retina and use a fine tip brush dipped in PBS to lay it flat GCL side down in the clover-like shape on a small square cut from nitrocellulose filter paper.

Pick up the corner of the nitrocellulose paper with forceps and place it in a 48 well plate with 4%power formaldehyde for one hour, then transfer the filter paper and retina to a well with PBS and wash three times for five minutes each. Thaw A4P0 solution on ice. Then transfer the retina into the A4P0 solution and incubate overnight at four degrees Celsius with gentle agitation.

Add vegetable oil into the well to completely cover the A4P0 solution. Incubate in a water bath at 40 degrees Celsius for three hours with no shaking. Then wash three times in PBS for five minutes per wash.

Incubate the retina in 10%Sodium dodecyl sulfate at 40 degrees Celsius for two days with gentle shaking then transfer the filter paper and retina to PBS with Triton X-100 and wash five times for 90 minutes per wash. After the final wash, store the retina at four degrees Celsius in PBS-T with 0.01%sodium azide or move directly to immuno staining. Remove the retina from the filter paper by gently peeling it off with a fine tip brush in PBS-T.

Incubated in primary antibody diluted in blocking solution for two days at 40 degrees Celsius with gentle shaking. After the incubation, wash five times for 90 minutes in PBS-T. Incubate the retina with appropriate secondary antibodies diluted in blocking solution for two days at 40 degrees Celsius with gentle shaking.

Protect the samples from light throughout the remainder of the procedure. Wash the retina five times for 90 minutes in 0.02 M phosphate buffer. Finally incubate the retina in sorbitol-based refractive index matching solution at 40 degrees Celsius overnight with gentle shaking.

Outline a glass cover slip with a fine tip permanent marker to mark a square boundary on the back of a glass microscope slide. Flip the slide over and use a syringe to trace the boundary with a thin line of silicone grease on the front of the slide. Leave a small gap in one corner for excess mounting solution to escape.

Transfer the retina to the center of the bounded area and position it with a fine tip brush so that it lies flat with the photoreceptor side against the glass slide. Pipette approximately 60 microliters of sRIMS so that it covers the flattened retina and extends to one corner of the enclosure. Ensure that the retina stays flat and in place.

Apply the cover slip, starting from the corner with the sRIMS and slowly lower it until it touches the grease on all sides. Place a stack of three cover slips on each side of the mounted retina as a spacer. Use the long edge of another slide to press down the cover slip so that the Mount is flat and even.

Store the slides at four degrees Celsius until imaging. Begin by placing the slide on the microscope stage and locating the sample. To obtain Z stacked images of samples, first focus on the signal in each channel individually and set the exposure time or scanning speed for fluorescence or confocal microscopes respectively.

Set the range for the Z stack either by manually setting the focal plane at the top and bottom of the desired range or by setting the midpoint and then specifying a range around the midpoint. Adjust the step size or number of slices as desired. Capture the image and save the original file.

Then exported as a TIF file or another desired format. Use image analysis, software of choice to adjust the brightness and contrast in each channel until optimum clarity is achieved in both the single images and the three dimensional rendering of the Z stack. When processed with the modified CLARITY protocol complete optical transparency throughout the thickness of the retina was observed compared to non-processed control retinas.

Z stacked images for Arrestin labeled cones in ONL, TH labeled DAC's in INL, and RBPMS marked RGCs in GCL are shown here. The relative location of neurons throughout the entire thickness of the retina was observed in the overlay image. TH staining and CLARITY processed whole Mount retinas was compared with images obtained from standard preparation.

Dendrites and axon-like processes of DACs were more clearly revealed in a clarity processed retina than in a standard retina. Axon-like processes of DACs exhibited complete ring-like structures in a clarity retina compared to a standard retina. Ring-like structures of CLARITY retina taken using fluorescence microscopy were almost identical to those observed using confocal microscopy.

The axon-like processes also ran toward the outer retina. Immuno staining against GluA2 and PSD-95 showed distinct puncta, revealing individual GluA2 containing AMPA receptors and putative post synaptic sites, respectively. An overlay image showed some puncta on DAC processes.

The points of putative co-localization were presented in XZ, YZ, and XY planes and TH co-localized clearly with both GluA2 and PSD-95. It's important that the retina remains flat during the hydrogel polymerization process so that the cleared retina can lay flat for the mounting and imaging process.