Ex Vivoイメージング実験アダルト ゼブラフィッシュから準備新鮮な網膜スライス

The overall goal of this procedure is to prepare fresh slices of Zebrafish retinas for experiments that require live imaging of fluorescent proteins or biosensors. This method can help answer key questions in the field of vision research, such as the physiological and metabolic roles of calcium ions in specific cell types and cellular compartments within a retina. The main advantage of this technique is that it provides spatial and temporal information about key metabolites and signaling molecules in live tissues.

To make it possible to remove the retinal pigment epithelium, or RPE, from the Zebrafish, dark adapt the fish for one hour prior to making tissue slices. For a slicing chamber, paint lines of nail polish onto a slide. Using clear nail polish, paint narrow lines to create a rectangle.

Repaint the polish once when dry. If the chamber will be used for static imaging, or injection experiments with minimal solution flow, then use a syringe to apply two parallel strips of petroleum jelly to a coverslip, one centimeter long and 0.5 centimeters apart. Now prepare the blades.

First, clean a double-edged razor with ethanol and allow it to air-dry. Then use scissors to cut it into approximately one centimeter pieces. Next, place the slicing chamber on the stage of the tissue slicer, center it horizontally on the stage, and mark the long edge with permanent marker for alignment.

Then, load a piece of blade onto the slicer arm. Secure the blade, so the edge is over the middle of the slide and runs parallel with the slide surface, but does not touch the nail polish. Then lower the blade arm using a one quarter turn.

Now, try slicing filter paper. Remount the blade if needed. To proceed, using the syringe deposit a single, small dot of jelly in a 10 centimeter dish, about 1.5 centimeters from the midpoint.

Then press the dry side of a coverslip into the jelly using forceps. Next, put another small dot of jelly about one centimeter from the inlet edge of the imaging chamber. Now, use a 20 gauge needle to make two one centimeter long thin parallel strips of petroleum jelly lengthwise along the center of the slicing chamber about one centimeter apart.

Then transfer the Zebrafish to a dish and cervically dislocate the fish without decapitation. Now, use a wire loop to loosen the connective tissue around one eye and then pull the eye forward gently. Next, carefully cut the white optic nerve under the eye using microscissors, but do not cut the back of the eye.

To proceed, transfer the eye to a dish of cold Ringer's solution on ice. Then harvest the other eye. Continue working under red light until all of the RPE is removed.

Next, transfer one eye into a drop of cold Ringer's solution on a glass slide on a petri dish. Then place the dish under a low-powered dissection microscope to dissect the eye cup under red light. First, pierce the cornea with fine forceps.

Next, use forceps and scissors to gently remove pieces of clear brittle cornea and silvery sclera. Then remove and discard the lens, and most of the sclera to isolate the eye cup. With minimal handling remove any pieces of fat and sclera that remain attached to the eye cup without removing the retina.

If the retina separates from the RPE, then proceed with extra caution to avoid damaging the delicate retina. Now, reposition the eye cup with open side down and cut it into thirds or quarters with a new blade. Discard the pieces of eye cup that are too curved to flatten out.

These pieces are often too small to be useful in any case. To mount the retina sections, wet a piece of filter paper with Ringer's solution and use flat forceps to place it next to the eye cup pieces. Then immerse the filter paper and tissues in cold Ringer's solution.

Next, use forceps to position retina pieces on top of filter paper with the RPE and photoreceptors up. Gently handle the edges of the eye cup pieces with fine forceps to orient them into a single line at the center of the filter paper. Now flatten the retina by using forceps to lift the filter paper with retina attached and setting it on a dry paper towel.

For about three seconds, let the force of liquid wicking into the paper towel flatten the retina. Process all of the retina pieces in this manner, just don't let them dry out. Now, apply a drop of Ringer's solution and use fine forceps to peel away any remaining black sheets of RPE from the tissue.

Peel gently, and if the retina lifts off the paper, wick away more solution as before and it should flatten again. Now transfer the paper with retina pieces to a slide and trim the paper into a rectangle leaving about 0.5 centimeters on either side of the row of tissue. Then, move the filter paper to the prepared slicing chamber.

Push the long filter paper edges into the thin petroleum jelly lines using forceps, and then immerse the retinas in three to four drops of cold Ringer's solution. First, transfer the preparation to the tissue slicer stage. Position the long edge along the marked line and secure the chamber ends to the stage with laboratory tape.

Then, starting at one end, cut the retina and filter paper using firm, gentle pressure on the slicing arm. Check that the first slice was cut fully, and then proceed to use the micrometer to cut slices that are approximately 400 microns thick. Next, transfer the chamber with sliced sections to the petri dish containing the prepared coverslip, which will serve as a imaging ladder.

Flood the dish with cold Ringer's solution and place it on the stage of the dissecting microscope. Now, under low magnification, use the forceps to secure a strip and move it onto the imaging ladder by sliding the underlying petri dish. Rotate the slices 90 degrees and bury the filter paper edges into the jelly.

It is important to keep the retina submerged and to avoid making any direct contact with the retina. Next, reposition the slices in the ladders so that the retina layers are clearly visible. For the slices at each end of the ladder, make the retina face inward toward the other slices to minimize motion during injection or flow.

Discard any slices that do not adhere well to the paper. Now, stain the tissue. Meanwhile, prepare the imaging chamber and injection apparatus.

First, flush the tubing with Ringer's solution, until no air bubbles remain. Then, attach the tubing to the imaging chamber inlet and close the stop cock. Then, refill the syringe with the solution for injection and reattach it to the tubing.

Now, use forceps to transfer the prepared imaging ladder to the imaging chamber. Press it into the dot of jelly near the inlet edge and fill the chamber with Ringer's solution to cover the slices. Then proceed with imaging.

Using the described techniques, perfectly transversed slices can be imaged through all of the retinal layers. When slightly rotated, bundles of the outer segments will be visible. Using PI staining, dead cells can be eliminated from analysis.

Healthy PI-negative photoreceptors have a stereotypical polarized elongated morphology. When in oxygenated Ringer's, they can be imaged for up to four hours. One imaging strategy is to visualize the dynamics of cone photoreceptor endoplasmic reticulum relative to the nucleus.

Using tissues that express GFP, and the cone ER, while counterstaining with a nuclear dye. A similar strategy can be employed to view the actin cytoskeleton with tissue expressing GFP-fused actin. Using time-lapsed imaging, cellular dynamics such as cytosolic calcium fluctuations in a cone photoreceptor can be observed.

For example, while isotonically depleting sodium in the imaging chamber, GCaMP can be used to visualize rising cytosolic calcium concentrations. By quantifying the fluorescence responses from single cone outer segments, cell bodies and synapses, the intracellular calcium dynamics can be determined during pharmacological manipulations and so forth. While attempting this procedure, it's important to remember to use red light before the RPE is removed.

Carefully flatten eye cups before slicing, and keep the slices submerged at all times. Once mastered, tissue dissection, slicing, and preparation for imaging can be done in less than 30 minutes. After watching this video, you should have a good understanding of how to prepare retinal slices from Zebrafish to study metabolism, and physiology in specific cell types in cellular compartments.