Adults.Debra Fish has a unique ability to regenerate neural system after injury such as hair cells, olfactory valve, T sland, spinal cord, and optic nerve. As the part of CNS optic nerve injury could induce RGC survival and degeneration. However, HG staining and antibody IHC could not specifically mark rrg disease with transgenic line marked all R GCs has not been constructed.
In order to research the change of R GCs number after optic nerve injury in adult zebrafish, we successfully labeled all RGC by retrograde labeling RGC from the tum. Hi, I'm I'm, we are members of Laboratory Of Neuro Development and Repair School of Lexus University of Sales and Technology of China. And today we'll show you how to retrograde labeling RG CS in ABRA fish and get RG CS number in whole mountain retina.
So Let's get it started. First, We need to build up A perfusion system to peruse anesthesia and the fresh water. You can find more details about the operators in the attached PDF document of this movie.
Put the box above and make sure the fluid can flow smoothly from the box to the fish. Keep all operators disinfected during the Surgery. Prepare several speedballs and put them in the caves.
Drip them with 75%ethanol and absolute ethanol. Prepare a neurore eye using a glass needle to deep it besides need geo Form acid etching, light curing bound tying forceps, jewelry forceps, acupuncture, ping blunt indeed forceps, scle rectum, scissors, coum swab. Salient blue LED glass needle with neurore eye on the end lighter and a 75%Ethanol.
Anize the fish with 0.03%MS triple two. Then put it into the gap of the sponge. Add just the velocity of perfusion to about one drop per second.
Put The needle into the fish mouth for oxygenation. Cover the fish with a piece of carpas to keep the skin wet. Now we turn to the top view for more details.
First, remove the skin over the Whole sc. Wipe the surface with coum swab. Wash thecal with salient.
Then dry it with ear washing bulb. Corrode the skull with the agent for 10 seconds, completely wipe away the agent. Then wash the area with salient and dry it Cover Areas.
Accept the right tectum with light cure inbound for protection towards the incoming corrosion. Cure it with a period of 40 seconds exposure to blue light. Put agent on right Tum again for completely edge.
For two minutes, completely wipe away the agent. Then wash the area with salient and dry it. Carefully open the skull with number five forceps.
To expose the right tactile, absorb the cerebral spinal fluid infusing out from the wound with geo formm. Coat the dye over the whole right tectum. Then cover a piece of geo formm on D eye to attach it to the whole right tectum Tally.
Cover the hole with the ster. Scale from the sand fish. Seal the wound with the light curing bound and Fix it with blue light.
Switch off the anesthesia Solution. Revive the fish with fresh water. Usually we cut off their Things so they won't swing fast.
In our lab, all surgeries are done by a skilled person. It takes 10 to 12 minutes per fish. About 20 to 30 fish can be labeled in half a day.
Retina is not included in the Celia Zone. Make eye cup by removing the Celia zone and cornea with venous Scissors. Remove lens, then flush out aqueous fluid with I-C-P-B-S.
Flush out pigment cut optic nerve at the disc. Remove The sclera to get a retina. Fixing the retina in 2%of PFA is optional.
If imaging is not quickly following the separation, Clean the blood clouds and the pigment fragments before mounting. Transport The retina with a spoon made from curved cover glass. The ages are marked by lines.
Put the retina In ic. 30%glycerin on a microscope.Slide. Keep RGCL Upward.
Remove the glycerine, then cut the retina into four quadrants. Drop 20 microliter. I see 75%glycerin on the retina.
Cover the sample with a square cover glass with a silence of 1.8 centimeter. Seal the cover glass with nail oil and the market. Observe the sample on fluorescence microscope via this Surgery.
We successfully retrograde labeling RTCs in. Adapt zebra fish and get the RTCs number in whole mountain retina. By using light curing bound, we improve zebrafish survival rate that's making this method More effective.
