I have been using a procedure for mouse eye fixation in paraformaldehyde. We remove the mouse eye after sacrafice. We immediately put it in 4% pfa for 30 minutes. After that we punch a hole in the eye, we do not remove the cornea or lens. After fixing for 4 hours, we put it in 10% sucrose for an hour, 20% sucrose for an hour, then 30% sucrose overnight. We then put the fixed eye into a cryomold filled with OCT mixture without sucrose. We freeze the tissue on dry ice. Sometimes the retina is perfectly sectioned, other times, it is extremely detached and has cells missing or shrunken. Any advice would be helpful. Thanks
Hi Matthew,
After 30% sucrose overnight, use a filter paper to absorb the sucrose as much as possible, then dip into OCT, remove the air bubbles in OCT, freeze the tissue by ethanol-dry ice.
You can extend the fixation time to overnight, if you want to keep the morphology better. However, longer fixation may reduce the sensitivity of your antigen -antibody reaction of immunohistochemistry or immunofluorescence. You need to find a fixation time point that is good for both your morphology and antigen keeping. I think 4%PFA fixation overnight with gentle shaking maybe suitable for both.
Good luck!
Mei Li
Be very careful when taking the eyes out. Sometimes just a little too much pressure and the retina will detach and no matter what you do after it is too late.
Hi Matthew,
I had best results with directly freezing eyes in OCT, and fixing them later as cryosections on the slide for 5 min. Hope this will help
Greetings from Germany
Hi Matthew,
Take a look of the enclosed paper, I hope it helps!!
the retina can very easily break at the level of the external segments of the photoreceptors. If that is where your observations will be then you could consider other methods (parafin or resin embedding). Anyway for frozen sections, you should very carefully remove the eyes, the rule is not touch/hold the eye directly - hold it through extraocular muscles or conjunctive tissue surrounding the eyeball. Put it in a small petri dish in cold PBS or 4% paraformaldehyde, then under a binocular make a hole through the cornea using a syringe needle (23G or 20G): hold the eye with fine forceps , orient it so the cornea is facing up then just puncture the cornea with the needle, making a neat and large cut. The needle has to be new - it has to go very easily through the cornea. The purpose of this is for fixative and later sucrose to get access quickly to the retina. Then fix the eye - for imunofluorescence staining 30 minutes fix time is best, put it on a rocker table in the cold room. Then take it through ascending sucrose-PBS (10/20/30%). Normally 1 hour each is sufficient. In between the fisx and sucrose you can store the eyes in PBS at 4°C. For embedding do as already suggested by Mei Li.
Good luck.
As soon as you 'punch a hole' in the eye you detatch the retina !
Leave the whole eye in 4% PFA pH 7.4 for at least 4 hours at room temp or overnight at 4 degrees then cryopreserve in sucrose until it sinks
After sucrose 30% I get the eyes all shriveled up. I reasoned it happened because the high osmolarity of the solution, and we even dissolve it in PBS.
How do you remove sucrose from the inner chamber of the eye? I've had to remove the lens to be sure that it was really entering.
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