Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Transcript

Mouse retinal dissection is a really delicate process due to the size and shape of the eyes and the fragility of the retina section. Imperative to practice, having a detailed protocol that provide efficient method and tips is the key to high quality retinal dissection and section. We provide tips and advice to help researcher avoid common pitfall and obtain some high quality retinal sections suitable for immunohistochemistry.

Mark the temporal part of the eye of a euthanized mouse using a tail cauterizer by touching the cornea very lightly and for no more than a split second to slightly burn the cornea and avoid making a hole. Immediately after that used curved Dumont 545 forceps to enucleate mouse eyes. Place the eyes in a 1.5 milliliter cryotube with one milliliter of 4%PFA and PBS and incubate for 15 minutes on ice.

Then, transfer the fixed eyes using the curved Dumont 545 forceps to a modified 35 millimeter dissection dish filled with PBS and place under dissecting microscope. Use a microknife to make a small incision at the burn mark perpendicular to and just above the limbus border of the cornea. Insert curved scissors into the incision and perform a circumferential cut of the cornea following the limbus.

Then, using thin Dumont 5 forceps, remove the cornea, extract the lens, and lift it delicately away from the posterior portion of the eye. The vitreous body will come out with the lens and the retina will be visible as a white surface covering the inside of the posterior eye cup. Wash the isolated eye cups twice in one milliliter of PBS for 10 minutes at room temperature.

To cryo-protect the eye cups, equilibrate them in 15%sucrose and PBS overnight at four degrees Celsius until they sink. Then transfer the eye cups to 30%sucrose and PBS for two to three hours until they sink. Place the eye cups in OCT in 10 by 10 millimeter cryo-molds for embedding.

Under a dissecting microscope, orient the eye in the cryo-mold along its dorsal ventral axis by rotating the eye until the burn mark is on top. The optic nerve is one the left and the cut out part of the mold faces the right. To snap freeze the retinal tissue, immerse the cryo-mold for at least five minutes in a metal beaker containing isopentane and then place the beaker in liquid nitrogen up to one third of the height of the beaker.

Remove the frozen block, wrap it in aluminum foil, and store at minus 80 degrees Celsius. Use a pen or Sharpie to mark the frozen block to record its orientation. After adjusting the cryostat temperature between minus 20 and minus 25 degrees Celsius, place the molds containing the embedded eye cups inside and allow them to equilibrate to the cryostat temperature for one hour.

Install the block in the cryostat with the dorsal ventral orientation and start cutting 10 micrometer thick serial sections. Carefully place the retinal sections on annotated and numbered microscope slides and then store in slide boxes at minus 20 degrees Celsius or minus 80 degrees Celsius until ready for immunostaining. Immunohistochemistry with antibodies to rhodopsin, a photoreceptor marker, glutamic acid decarboxylase 65, and amacrine cell marker, glutamine synthetase and Muller cell marker, and calbindin, a horizontal cell marker was performed on retinal sections.

The results indicate that this protocol yields high quality and well preserved retinal sections unlike in poor quality retinal sections obtained from a different protocol. Rhodopsin rich inner and outer segments remain vertical and intact with little to no separation from overlying the retinal pigmented epithelium. Interneurons, such as Gad65 positive amacrine cells, remain properly stratified within the inner nuclear layer and inner plexiform layer.

Also, Muller cells remain well preserved with soma properly aligned in cytoplasmic processes that span from the ganglion cell layer to the outer nuclear layer. Furthermore, well defined horizontal cells are detectable at the inner nuclear layer and outer plexiform layer border. It is important to mark the topper region of the eyes and keep the orientation during embedding.

Always prepare paraformaldehyde under the hood with the appropriate personal protective equipment.

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Journal (Neuroscience)

Journal (Neuroscience)

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

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