Cryosectioning and Immunostaining of Mouse Retina

Our research focuses on the study of retinal diseases. The question we are trying to answer are the molecular mechanism underlying retina degeneration. Immunostaining of mouse retina sections and high resolution imaging are the key technologies used to advance research in our field.

Consistently obtaining retina sections with good morphologists is one of the current experimental challenges many researchers. The significant findings we have established in our field, including identification of PDEdelta as a prenyl-binding protein, and UNC119 as an acyl-binding protein. To begin, place the sacrificed mouse on an operating table.

Using a blue marker, mark the superior side of the eyeball on the sclera. Remove the eyeballs using ophthalmic scissors. Transfer the dissected eyeballs to a 2-milliliter round bottom microcentrifuge tube containing one milliliter of 4%paraformaldehyde for 10 minutes.

After fixation, place the eyeballs in an inverted 6-centimeter Petri dish. Under a dissecting microscope, use fine forceps and ophthalmic scissors to make a small incision on the cornea. Place the eyeballs back into the 4%paraformaldehyde solution for an additional two hours of fixation on ice.

Next, remove the fixative from the tube and wash the eyeballs three times with PBS. Transfer the eyeballs to a 2-milliliter tube containing one milliliter of 30%sucrose solution for cryoprotection at four degrees Celsius overnight. The next day, transfer an eyeball onto an inverted Petri dish with the cornea side facing upward.

Blot the excess solution from the cornea using a lint-free wipe. Dip a two to three centimeter segment of tennis string into cyanoacrylate glue contained in a 0.2-milliliter microcentrifuge tube and remove it quickly. Under a dissecting microscope, attach one end of the tennis string with residual super glue to the center of the moist cornea.

Allow the glue to solidify for 10 to 20 seconds, then grasp the other end of the tennis string and dip the eyeball into a 200-microliter microcentrifuge tube filled with super glue for one second. Quickly remove the eyeball and immerse it in PBS. Blot the excess PBS from the surface of the solidified glue using a lint-free wipe.

Under a dissecting microscope, remove the cornea using ophthalmic scissors while holding the attached tennis string. Using forceps, carefully extract the lens from the eye cup. Transfer the eye cup to an embedding mold filled with the optimal cutting temperature or OCT compound.

Next, completely fill the eye cup with the OCT compound. Position the eye cup so that it faces the sidewall of the embedding mold, ensuring the sagittal plane is parallel to the bottom of the mold, using the blue mark on the sclera as a reference. Transfer the embedding mold containing the eye cup to a 80 degree Celsius freezer for five minutes.

Transfer the frozen eye cup to a cryostat set to 20 degrees Celsius and allow it to equilibrate within the chamber for 30 minutes. Section the eye cup at a thickness of 12 micrometers. Then mount the sections onto a positively-charged glass slide.

To begin, bake the frozen mouse eyeball sections on a slide in a 37 degree Celsius oven for 30 to 60 minutes. After baking, using a PAP pen, draw a circle around the sections on the slide. Immerse the slide into a Coplin jar containing PBS.

Place the Coplin jar on a slow shaker and wash the sections twice for 10 minutes each to remove the OCT compound. Next, place the slide horizontally in a moisture chamber. Add the blocking solution to the circled sections and allow blocking and permeabilization at room temperature for 30 minutes.

After blocking, replace the blocking solution with the primary antibody solution. Incubate the sections in the moisture chamber at four degrees Celsius overnight. Aspirate the primary antibody solution and wash the slide twice with PBS for 10 minutes each.

Then add the secondary antibody mixed with DAPI diluted in the blocking solution. Incubate the sections in a dark moisture chamber for one hour. After incubation, rinse the slide twice with PBS.

Using a Kimwipe, blot away any remaining PBS from the slide. Apply an appropriate amount of antifade mounting medium to the sections and cover them with a cover slip. Immunofluorescent images of mouse retinal sections showed that the photoreceptor outer segments were successfully labeled with anti PDE6B showing distinct and morphologically intact segments with no retinal detachment.

The inner segments and the outer nuclear layer were preserved, showing no structural damage.