事項をカットする場所: 郭清と眼のランドマークからマウス網膜の空間的なオリエンテーションの解析ガイド

The purpose of this dissection protocol is to provide a standardized methodology for the use of ocular anatomical landmarks, like the rectus muscles, choroid fissure and S-opsin gradient for the topographical orientation of the mouse retina. The main advantage of these techniques is that they provide a reliable way to accurately identify the dorsal, ventral, nasal, and temporal poles of an isolated mouse retina. Begin by identifying a point on the dorsal cornea near the cornea sclera border and between the nasal and temporal canthi.

Touch the cornea with a heated cautery pen for less than a second to create a burn mark for orientation purposes. Repeat for the second eye. The presence of the dorsal burn allows for identification of the superior rectus muscle.

For enucleation, use curved forceps to gently push the eye out of its socket and grip the globe from underneath. Then, slowly lift the globe from its socket while simultaneously moving it gently from left to right until the globe is released from the socket. Transfer the globe with the attached rectus muscles to a Petri dish containing dissection medium.

If working with both eyes, make sure to keep track of which eye is the left eye and which is the right eye. Under the dissection scope, visually locate the dorsal corneal burn and identify the superior rectus muscle with which it is associated. Using dissection scissors or a 20-gauge needle, puncture the cornea at the burn mark.

Then, make a deep relieving cut into the globe toward the optic nerve to bisect the superior muscle. An isolated retina with this cut is seen here, and reconstructed retina with this cut is shown here. Use two sets of forceps to begin to isolate the retina by gently tearing the hole made with the puncture until part of the retina is exposed.

Then, tease apart the retina from the sclera until the sclera has been completely removed. Remove the iris, lens, vitreous, and any remaining structures until the retina is completely isolated. The presence of a dorsal burn allows for the identification of the nasal and temporal choroid fissure.

If working with the right eye, the nasal choroid fissure is located to the right of the burn and the temporal choroid fissure is located to the left of the burn. This is reverse of working with the left eye. To use the choroid fissure landmark to identify retinal orientation, locate and identify the choroid fissure on the back of the eye.

Orient the globe so that the dorsal burn mark is located at the superior pole, as it would be if the eye were still in the mouse. Then, use dissection scissors or a 20-gauge needle to make one puncture in the globe, first where the dorsal burn is located. Next, make a shallow relieving cut toward the optic nerve where the dorsal corneal burn is located.

Then, make the following two deep relieving cuts toward the optic nerve. One by lining the blades of the dissection scissors up with the temporal choroid fissure line on the back of the eye, and one by lining the blades of the dissection scissors up with the nasal choroid fissure line on the back of the eye. These cuts are shown here on an isolated and reconstructed retina.

After making these cuts, use two sets of forceps to gently tear away at the puncture holes until part of the retina is exposed. Using dissection scissors, make four relieving cuts in the retina so that it will lie flat. Mount the retina, ganglion cell side up, on nitrocellulose membrane by gently pressing each corner of the retina onto the membrane with forceps.

Then, use forceps to immerse the mounted retina into one milliliter of 4%paraformaldehyde contained in the first well of a 24-well plate. Place the 24-well plate on an orbital shaker at room temperature and fix the retina for exactly 40 minutes. After fixing, transfer the retina to the second well of the plate, which is filled with one milliliter of 0.1 molar PBS and wash the retina for 15 minutes at room temperature.

When washing is complete, transfer the mounted retina to the fifth well containing one milliliter of blocking solution and incubate overnight at four degrees Celsius. The next day, add the rabbit anti-S-opsin primary antibody to the blocking solution at a concentration of one to 500 and incubate for three days at four degrees Celsius. After the three day incubation, wash the excess primary antibody from the retina six times by sequentially placing it in six wells filled with one milliliter PBS for 10 minutes each at room temperature.

Then place the washed retina in a well with fresh blocking solution and add donkey anti-rabbit Alexa 594 secondary antibody. Incubate the retina with the secondary antibody overnight at four degrees Celsius. Following the incubation, wash the retina in PBS six times, as before.

Using forceps, transfer the mounted retina to a Petri dish containing PBS. Release the retina from the nitrocellulose membrane by gently inserting the tips of forceps between the retina and the membrane until the retina is no longer attached. Immerse a glass microscope slide in the PBS and mount the retina on the slide by floating it onto the slide and gently prodding it with forceps until the retina sticks to the glass.

Cover the retina on the slide with mounting medium and place a 1.5 coverslip. Put the slide in a slide tray and allow it to sit at room temperature for an hour. After an hour, place the slide tray at four degrees Celsius.

After the slide has been coverslipped for 24 hours, use nail polish to seal the sides of the slide to prevent desiccation. This is an example of a retina isolated from the right eye that has been immunostained to label S-opsin and imaged with an epifluorescence microscope. It is important to note that the cuts in this retina are arbitrary since the topographical orientation can be identified by the S-opsin gradient.

The acquired image of the retina can then be digitally reconstructed with ReadiStruct software. The original cuts are pseudo-colored red in this image. Notice that the output file of this program does not correctly align the retina.

After the retina has been run through the custom mat lab code, the retina is rotated so that the highest concentration of S-opsin staining is placed at the bottom and identified as the ventral retina. This image shows the retina from a left eye. The temporal pole is located at 90 degrees counterclockwise from the ventral pole and the nasal pole is located 90 degrees clockwise from the ventral pole.

Once mastered, these dissection techniques can be done rather quickly in just a few minutes if performed properly. After watching this video, you should have a good understanding of how to accurately and reliably orient the mouse retina using the superior rectus muscle, choroid fissure, or the S-opsin gradient as a landmark.