We are facing problems with tissue preservation. We have both mechanical damage in the tissue may be resulting from handling and broken cell membranes indicating unproperly set osmolarity in the solutions we use.
Thank you for your answer and suggestions. We try to avoid perfusing the animals here in Italy because of the animal activists.
I am sorry, I cannot advise you on specifics of retina specimen preparation, but it looks like you problem is in wasting time on the whole eye fixation. Too big for successful fixation. You may want to dissect it as soon as possible, then fix it.
Probably true, this is exatly what we are planning to do now :) Thanks! :)
I managed to optimize our TEM protocol (see attachment) and it was an absolute success! I'd like to point out, that before primary fixation I prepared eye-cups removing the cornea, the iris and the lens; plus before secondary fixation I prepared eyecup quadrants and did NOT detache the retina from the sclera! It turned out to be an excellent protective shell....our degenerated retina is extremely fragile since there is no ONL left in the central area.
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