Hi,

One of the most common fixative for eyeball is the bouin's fixative. It gives a pretty nice fixation of all eyeball and preserve the retina.

hope this helps

Daniel

Thanks Daniel. But, do you mean the tissue will be fixed without first getting rid of the aqueous and vitreous humor inside it? What will happen to these fluid while the tissue is being fixed please? And on my second problem of how to section the specific parts of the retina, are there techniques/procedures widely accepted in doing this?

Yes this is what I meant. Dissecting out some eye parts could leads to disturb anatomy of the retina in particular. The most tricky part is to cut the lens that is really hard, so including in Bouin's soften it. You could still enucleate it but be gentle since you can damage the retina, then you could go for PFA fixation or so.

best

daniel

Thank you very much. Please what do I do in a case where am working on paraffin embedded sections. I don't have access to cryosectioning.

After PFA or Bouin's fixative just proceed to dehydradation through ethanol step and standard procedure for paraffin embedding.

Daniel

For paraffin section all you have to do is : once the eye is enucleated drop it in 4% PFA for 18 hours no less, then move to PBS until ready to cut. With this technique, there is no chance to damage or detach the retina (as long as you are careful not to pull on the eye ball when enucleating. For staining you can do antigen retrieval (microwave in Na citrate solution after deparaffinsing the sections. We had the best staining. If you need frozen section let me know. good luck. Aicha

I may be too late to answer... 10 % neutral buffered formalin is used by my colleagues to fix and keep their samples for paraffin embedding. Note, that while paraffin sections are generally better in retaining anatomical features, the immunohistochemsitry is worse/weaker compared to cryosections. Also note, that a long fixation in itself is not that great for IHC, usually one needs to fix "just enough" to get good IHC.

Although the mouse eye is small, we find that it is generally a good idea to poke a little hole in it so that the fixative has better/quicker access to the retina. We usually use a fine needle and penetrate on the edge of the cornea.

In case you end up doing cryosections: it's worth removing the anterior half (cornea, lens) after an initial PFA treatment. So the "protocol" (for cryosections) would be: take out the eye, clean off excess tissue, poke a hole, drop in 4 % PFA for 10 minutes, take out and remove anterior portion, drop the eyecup in fresh 4 % PFA for one hour (max), take out, wash with PBS, cryprotect and embed. (You'll need a stereomicroscope for the "poking" and for the removal of the anterior part.)

Thanks very much. This contributes to my knowledge and I shall try it out.

For paraffin sections, eyes should be fixed in Davidson's Fixative.

Glacial Acetic Acid 100 ml

95% ethyl alcohol 300 ml

10% Neutral Buffered formalin 200 ml

Distilled or Deionized Water 300 ml

The acetic acid softens the lens which allows for paraffin sectioning.

Fix for a day and transfer to 70% EtOH. You can process it normally after that.

Hi, you can place the whole eye in Davidsons fixative as above, you can use a microtome blade to cut the eye. The attached file may be useful

Hello Ahmed, In addition to what has been said, please look at my recent publication Am J Pathol. 2014 Sep;184(9):2403-19.

In all the images presented in this work,

for frozen sections, after taking the eye balls, the eyes were immersed in 4% PFA for 15 min, then under dissecting microscope make very tiny small incision at the level of the limbs (this will allow prefer fixation), then put the eye balls back to 4% PFA for a few hours. Transfer he eyes to 1% PFA until cutting. If you do this gentle enough will have perfect morphology).

For paraffin section, drop the eye balls in 4% PFA for at least 18hrs and up to 24hrs, then transfer to PBS. As you can see form my histology images the structures of the retina are perfectly preserved.

If you are interested in the  cornea you could do fresh frozen technique which is the best for this part of the eye. Hope this is helpful and good luck. Aicha