Hi, I am Tu from GOSO Lab in the University of Hong Kong. Today I'm going to introduce how to retro grid label rat. Retinal gang cell.
Retinal gang cell will be retro grid label by applying forensic dye for go on the surface of superior click so that later on we can count the retinal ganglia cell on the flat prepared retina to evaluate the cell death or the effect of drug applied. This is a photo illustrate zero pathway from the retina to the superior. CLE fluorescent thigh can be retro greatly transported from the superior CLE through the optic tract.
Cross the optic chama through the optic nerve to the Retina in the eye. We use operating microscope to do the Surgery. Welcome pump for aspirating the cerebral cortex over the dorsal surface of CLE head stage for holding the red hat in position.
Ke and sine for izing rat driller for opening the school and absorbable jealousy pon for applying fluorescent dye FG on the surface of clickless. The instruments were autoclave before operation, including fine forceps, spring scissor, vacuum needle, drill tip and suture clips. Surgical blade for cutting the skin, ruler for the measurement and cotton are also in sterilized condition.
We analysis The red by tonal injection of ketamine and sine gel. After shaving the adver from the level of eye to the level of years, we disinfected operation area with propane odine, followed by 70%alcohol. Then put the red hat in position on the head stage.
Cut the skin open in the middle line of the head, starting from the level of eye to the level of ears. Then we have a good exposure of chrono suture, sagittal suture, transport suture, and this is the BMA and this is the Lambda. Mark the screw at 0.5 to one millimeter from AL end transfer suture and then label them on both side mirror again to make sure that the mark is at least 0.5 millimeter away from the sutures.
Then we can start to drill a hole along the marker side, control the speed and direction of the driller to a wide rupture of meninges and sinus lining beneath the suture. Stop Drilling when the diameter is about two millimeters. And then we start the other Side mirror Again to make sure that the hole is at the right size and in the right place.
Cut the meninges open by the spring scissors, Use fine forcep to clean the debris of bone in the hole. Now We can Start the aspiration using the vacuum pump. The first layer of the BR is the green matter cut with the meninges again.
Then you can see that the tcell color changing to white. So these are the white matter of the sero. Do the aspiration nice and slowly.
So now we can see the gray matters again. The color is changing less white. You can see it is gray at the bottom.
I see. So you can see there the clear CSF is coming out. That means we are reaching the dorsal surface of the superior corus.
It's again, enlarged hole at this level. And then at the bottom we can see that the yellowish color dura covering the superior clus. You do the horizontal aspiration at this level to avoid destroy the content of superior clus.
Then we can start the as a whole. You see the green matter. It's actually green color.
And then we come to the white matter. The tissue color is white. Now you can see the CSF is coming beneath that gray color so nicely.
Remove that last layer of gray matter and then the yellow dura is exposed. Careful now to disrupt the dura over the separate clickers and then enlarge the hole. Try to make sure the full edge of the clickers can be observed under the microscope.
You Take The head clamp of the head stage at different angles and give some final aspiration until the four edges of the CLE can be observed directly under the microscope. Absorb The fluid in the hole with fine cotton strip to make the surface of CLE as strong as possible. Take a piece Of gelatin, ponty that pre silked with 6%fuel gold and separate it into eco pieces.
Make it into a thin layer and the size is a little bit louder than the entire dorsal surface of the superior. Carefully put it on the surface of the supreme clickers, and then the opposite side. Put it inside the hole covering the entire surface of the clus Through the holes with more jetting waning.
Finally, close the skin Wound with suture clips and clean the wound after surgery, put the red back into a clean cage and allow it to recover. And the warm condition. The red is behaving normally after operation while awake.
Seven days after SC labeling, we dissect the retina and flat mounted, then count the retinal gang cell and the epinal microscope using UV filter. Since 98%of retinal gang cell project to the superior clickers, a reliable ST labeling is fundamental for the evaluation of disease progression and drug effect. This is an example of retro labeled retinal gang cell on a fat mounted retina.
