Begin by immersing the split mouse retina with attached nitrocellulose membranes in 4%paraformaldehyde on ice for 30 minutes. Next, replace the paraformaldehyde with 5 to 10 milliliters of room temperature PBS to wash the split retinas. Before removing the split retina from the nitrocellulose, use a hydrophobic barrier pen to prepare circular wells on a microscope slide.
Let the wells air dry for 5 to 10 minutes. Afterward, place the split retinas in the prepared wells and add enough PBS to immerse them. Under a dissection microscope, gently lift the edges of the tissue with a fine paintbrush and circle around the retina to separate it from the membrane.
Use forceps and the paintbrush to remove the membrane from beneath the floating piece of the retina. Then carefully aspirate away the remaining PBS, so that the retinal tissue rests on the microscope slide ganglion cell side down. Immunofluorescence labeling of synaptophysin across the top of the split retina indicated that the photoreceptor synaptic terminals were retained.
However, photoreceptor nuclei were absent, confirming the retina split through the photoreceptor axons. Synaptic integrity was assessed by labeling RGS11 in ON-BC dendrites and CtBP2 in photoreceptor synaptic ribbons. Splitting did not damage the morphology of photoreceptor terminals in the outer plexiform layer.
RBC viability in the split retina was assessed using a near infrared nuclear dye. Regional variability in cell viability across the tissue was observed with higher cell death in some areas with most RBCs remaining viable after splitting. Immunofluorescence labeling of RBCs against PKC alpha and horizontal cells against Calbindin-D showed rapid antibody diffusion in the split retina after one hour incubation.
