En face Cryosectioning of Mouse Retina for High-dimensional Spatial Molecular Analysis

The retina is a useful model for understanding neuronal computations. These computations depend on the special positions of cells. However, comprehensive maps of this have remained elusive.

Over the last decade, single cell sequencing provided clear descriptions of retinal cell types. Now, spatial transcriptomics provides a novel way to use these descriptions in intact tissue. A critical challenge for using spatial transcriptomics is that they require very thin tissue sections. The retina is already quite thin in the horizontal plane we're interested in.

By inventing this method to create thin and fast sections, to use spatial transcriptomics in machine learning to map all retinal ganglion cells in the same tissue. Over the past 30 years, only 17 out of 45 mouses' retinal ganglion cells were mapped, and this technique enabled us to map all of them at once.

[Instructor] To begin place a euthanized mouse on the work table. Ran the temporal pole of the globe on the cornea to maintain orientation and create a weak point for dissection. Submerge the globe and aim solution in a dish containing a piece of filter paper. With sharp number five forceps and Iris scissors carefully remove all musculature, fascia, vasculature, and nerves from the globe without puncturing the sclera. Position the tip of one Iris scissor blade perpendicular to the corneal brand. Puncture the globe at this position. Make a single clean incision through the cornea and limbus, continuing into the sclera and retina toward the optic nerve head. Then position the blade perpendicular to the initial cut, approximately one millimeter posterior to the limbus. Begin cutting radially through the sclera and retina. Maintain a consistent cutting line posterior and parallel to the limbus by using sharp forceps to guide and rotate the globe. If the retina and sclera separate, reposition the Iris scissor blade to cut them together. Estimate the one millimeter posterior distance using the middle width of the Iris scissor blade After completing the radial cut parallel to the limbus, inspect for remaining retinal connections. Carefully sever these with Iris scissors until the anterior and posterior globe sections are separate. Next, use two pairs of sharp number five forceps to gently separate the anterior and posterior portions of the globe. In older mice, if transparent fibers connect the lens to the retinal cup, carefully sever these with Iris scissors while minimizing retinal scleral separation. Using the filter paper as a support platform, cut the retina into a clover leaf, also called an iron cross pattern with Iris scissors. Repeat the dissection with the second eye. Cut the tip of a disposable plastic transfer pipette to create an opening larger than the dissected retina and sclera. Gently suction the retina into the pipette and deposit it onto a glass microscope slide. With a laboratory wipe, remove excess aim solution until the retina no longer floats and can be flattened. Now use two sharp number five forceps to unfold the retina and position it flat with the inner eye cup facing upward. Make relief cuts 0.5 to two millimeters long in each clover relief segment to aid flattening and adjust to prevent folding. Remove any remaining aim solution except for the liquid adjacent to the retina. Using a cover slip, quickly press and transfer the retina so the outer sclera faces upward and the inner eye cup is in contact with the cover slip. Verify that the retina is not folded and make adjustments if needed. Add aim solution to assist manipulation, but remove excess with a wipe to prevent curling and allow air drying for five to 10 seconds. Now apply OCT medium to cover the retina. Then use forceps to evenly spread the layer across the cover slip to prevent flotation. Flash freeze the retina in OCT medium using a metal plate cooled with liquid nitrogen vapor, or by contacting dry ice directly. Store the frozen retina is on dry ice for immediate use. For cryosectioning, create an OCT dome on the specimen chuck, occupying the inner two to three annuli if using the same cryostat model. Align the specimen chuck flat to the cutting surface. Create or note the orientation guide relating the chuck to the cryostat stage. Then trim the dome to form a flat surface parallel to the blade. With a razor blade remove excess OCT laterally and carefully separate the retina from the cover slip to make it mobile. Remove the chuck from the stage and apply a small drop of liquid OCT to the prepared flat OCT surface. Next, place the retina directly onto the liquid OCT. Use a pre-chilled glass slide to press and hold the retina until frozen. Apply more OCT over the retina and chuck to ensure bonding. Then freeze the assembly in the cryostat rapid freezing area. For sectioning, carefully trim excess OCT until the retina is visible. Use a razor blade to shape the OCT block edges close to the retina. Continue trimming while inspecting for sclera presence. When the sclera is visible, adjust stage angle as needed. Stop trimming when the sclera is approximately evenly distributed across sections. Now, use pre-chilled slides and two paint brushes to manipulate the sections and collect them. Place a finger under the section for about five seconds until the OCT melts and adheres the tissue to the slide. Then return the slide to a cold surface for refreezing. Store the prepared slides on dry ice for immediate use. Distinct Immunoreactivity or RBPMS in the ganglion cell layer and clear separation of vascular plexi labeled by tomato lectin confirmed successful preservation of retinal lamination and protein markers. Multiplex RNA detection using a 12-plex RNA Scope panel successfully maintained spatial structure and revealed distinct expression of markers such as Grm6 and RBPMS across the on face slice. High resolution images confirmed single cell expression patterns for RBPMS and other markers like Mafb6 and Meis2, validating both protein and RNA localization in the ganglion cell layer. Uniform signal distribution across clover leave sections and strong signals in xenium spatial transcriptomic data confirmed successful high-throughput profiling and RNA integrity. Single cell resolution visualization using xenium matched manual detection patterns confirming the protocol's robustness across platforms. Cell type specific transcript markers showed stratified laminar distribution in retinal cross-section and on face mapping, preserved their location across depths. Detailed zoom into the ganglion cell layer confirmed accurate spatial transcript detection relative to DAPI nuclear staining.