This post is mostly for the benefit of Andrea (*wave!*) who wanted to know how it could be done. It will be pretty gory, so for those who don't want to know how I cull rats and remove their eyes....it might be better if you wait for the next post. Cheers!
Ok.
Last chance!!!
Firstly, the rat is not killed by cervical dislocation (thank god cos theyre huge! I also have a story about this concerning Lewnotab). It's injected with ketamine to put it to sleep. When the rat is completely out, you squeeze around the orbital bones with the scissors so that the eye protudes. The blades are then worked underneath the eye orbit and sever the optic nerve. After both eyes are removed, the rat is killed with an injection of phenobarbitol.
Holding the eye gently, the cornea is removed from the eye by cutting just behind a white line that separates the front of the eye from the eye cup (I can't remember the technical name). The lens is removed and gently pull out the vitreous without detaching the retina. This whole bit is quite hard but once its done, you're set. The eye cup is fixed in paraform for 30 mins. It then goes through a series of phosphate buffer (NOT PBS) washes for 10 mins each. After the washes it is put through increasing concentrations (10%, 20%, 30%) sucrose for at least an hour each. At 30%, the eye cup is placed in the fridge for at least overnight.
To remove the retina from the eye cup...there's a colour difference between the two and if you can work the fine forceps between them, you can peel the retina off quite easily and there you go!!!
Hopefully I will soon have some lovely photos of the S-100beta, desmin and smooth muscle actin immunos that I've done on some vertical retina sections. Erica said they kicked ass!!! They're not perfect - too much background staining for my liking - but they're nice and fluorescent green and you can see the Muller Cell/astrocyte and smooth muscle morphology really nicely.
No comments:
Post a Comment