Dear Mohammad,

I work on rats and mice, but I can have a similar detachment problem with my sections unless I 1) remove the cornea and lens before embedding the tissue, and 2) section thru the eyecup towards the optic nerve.  That is, the eyecup passes over the blade first, and then thru the optic nerve.  But, I usually section they eyecup and the optic nerve, not just the retina and nerve head.

I do not know why the retina becomes detached when you add the OCT medium, however.  Are you cutting fresh frozen tissue, or fixed?  If you are using fixed tissue, do you use a stepwise sucrose gradient for cryoprotection?  I would try that solution first, by sequentially sinking in 5%, 10%, 20%, and 30% sucrose in 0.1 M Sorensen's phosphate buffer pH 7.4.

There is also cryo-gelatin embedding protocols.  This gives more support to the tissue that OCT, but your subsequent protocols have to be compatible with incubating the tissue at 37 C while it is infiltrated with the gelatin-sucrose mixture.  If you need the recipe, please let me know.

Good Luck!

Jill 

Dear Mohammad,

I work on rats and mice, but I can have a similar detachment problem with my sections unless I 1) remove the cornea and lens before embedding the tissue, and 2) section thru the eyecup towards the optic nerve.  That is, the eyecup passes over the blade first, and then thru the optic nerve.  But, I usually section they eyecup and the optic nerve, not just the retina and nerve head.

I do not know why the retina becomes detached when you add the OCT medium, however.  Are you cutting fresh frozen tissue, or fixed?  If you are using fixed tissue, do you use a stepwise sucrose gradient for cryoprotection?  I would try that solution first, by sequentially sinking in 5%, 10%, 20%, and 30% sucrose in 0.1 M Sorensen's phosphate buffer pH 7.4.

There is also cryo-gelatin embedding protocols.  This gives more support to the tissue that OCT, but your subsequent protocols have to be compatible with incubating the tissue at 37 C while it is infiltrated with the gelatin-sucrose mixture.  If you need the recipe, please let me know.

Good Luck!

Jill 

Dear M,

I work on mouse, rat and rabbit eye and like Jill M. said, you can have retina detachment if you remove lens before embedding in OCT compound.

Your problem can due to

-the way you remove lens: I remove cornea/ anterior chamber and after carefully the lens (if I rinse the eyeball after dissection I use PBS with Ca2+ and Mg2+).

-the cryostat temperature that is too cold when you cut the eye. I cut at -16°C and the result on unfix tissue in very good even if the lens is removed before.

Another solution is to fix your tissue before dissection of the eye and OCT embedding. However, some fixative liquide (like PFA and Bouin-Hollande) shrink the tissue and it make retina detachment, I think the better one is the Davidson solution but your subsequent protocol have to be compatible with this fixative...

Good luck.

VM

Hi Mohammad,

I agree with what have been said before and I have a few more suggestions:

First, I work with mouse and human tissue and that is a common problem with OCT embedded samples. What you can also try is fixing with Glutaraldehyde (that’s what we do for the TEM protocol when we really can’t have detaching, but if you want to do immunohistochemistry that won’t be compatible with this fix.

You can also try the Serra’s fixative (is formaldehyde, ethanol and acetic acid based) followed by an ethanol dehydration (that should potential make the chance of your retina to detach higher but that is not what we see in my lab. But once again this might not be compatible with what you want to do with your samples next..

Eye cupping is great to assure a good fixation of the layers but you need to be very careful because is super easy to promote the detaching itself during the eye cup procedure.  With human samples we usually pierce through the cornea and submerge them in formalin and let them fix for 12 to 24 hours and them we eye cup them. We don’t see detachment most of the times but we embed them in paraffin.

Good luck.

Joana

Dear Mohammad,

I second what Joana and Virginie say about carefully removing the lens!  That is probably the most difficut part of preparing the eye cup-the vitreous stays gooey, since it has so much carbohydrate.   I also puncture the mouse eye thru the cornea before putting it in liquid, even if I have fixed the tissue by transcardial perfusion.

Good Luck-

Jill 

Thanks everyone for your suggestions. Definitely it helps. However, I want to share my protocol briefly so that you can have better idea about my problem and suggest accordingly.

Yes, it would be better if we could fix the eye before the cornea and lens removal as it allow the retina to remain flat and attached to choroid but the problem is most of the time we receive the eye from eye bank without cornea and lens as cornea is kept for transplantation. So after receiving the eye or better to say eyecup, I fix it in 4% PFA for 2 hours before application of 10%, 20%, 30% sucrose solution for cryopreservation.

Another fact that I must share is, I don't embed the whole eye cup in OCT medium. My particular interest is ONH and the area surrounding the optic nerve head. So, I cut a rectangle shaped area (e.g. 12 mm X 8 mm) keeping the ONH in middle. Then I embed the tissue horizontally in OCT media facing the retina up. As soon as I place the tissue in the OCT medium the retina tend to lift off the choroid a little bit which I can't prevent. Then I do the snap freezing in Isopentane kept in dry ice.

In cryosectioning step, I use the object temperature -20 and section thickness 10 micron. After cryosectioning when I check the sections under microscope I see a huge gap between the layers (retina, RPE, choroid) and I think the gap is justified because it already had gap between retina and choroid during embedding process.

This is the details of my protocol and problem. I badly need to solve this problem. Therefore, I am looking forward to your suggestions. Thanks again for your concern and valuable feedback. 

Dear Mohammad

I have the same problem with mice eye, Has your problem been solved?

Dear Sara, Thanks for your query. My problem was not solved fully but I came up with some tricks to minimize the detachment of retina from choroid during processing. Actually slow freezing (instead of snap freezing) & keeping the retina in place with a fine needle during freezing helps a lot minimize the detachment. If you want you may try these and let me know if it helps. Best of luck.

Dear Mohammad,

When you place your sample into the OCT, can you place it horizontally, but retina first (I am not quite sure what you mean by horizontally)?  The OCT is pretty viscous, and as the tissue sample sinks there is a tendency for the retina to "float" away from the choroid.  If you reverse the direction, the OCT might push the retina towards the choroid.

That is actually one reason why I put OCT into my eye cups first, and then place it into more OCT in the embedding mold.  The other is that I want to replace the 30% sucrose trapped in the eye cup with OCT, so that the block is more uniform and sections well.  I like to freeze in molds because I have better control over orientation, since I embed entire eye cups + optic nerve.  I freeze on powdered dry ice, which may be your definition of slow freezing.

Just a thought.

Best of luck,

Jill 

I work with mice eyes. After the 30% sucrose incubation, I embed the eyes in a 37°C warm mixture of 7.5% gelatine 10% sucrose in 0.12M Phosphate buffer. This mixture gel at RT (you can accelerate the geling on ice) and is transparent but become white when frozen. Once embedded, you can freeze the eye.

Best

Kim

This is directed mostly toward Dr. Jill Miotke but I would welcome input from anyone:

I am wondering if you could share any tips regarding this answer:

"...That is actually one reason why I put OCT into my eye cups first, and then place it into more OCT in the embedding mold. The other is that I want to replace the 30% sucrose trapped in the eye cup with OCT, so that the block is more uniform and sections well."

I've been having a rough time getting quality OCT sections and I think it's because OCT is not getting fully into the eyecup (i.e. up against the retina where vitreous usually is). When I section the eyecup there is therefore an air bubble within the eyecup which can cause pretty severe crumpling/folding/tissue damage. Can you share how you get the OCT into the eyecup to displace the sucrose? I think doing that more effectively could be a huge help to me.

Thanks for your time.

Hi All,

I have similar issue and would appreciate your advice.

I have perfused mice with 1% PFA and post fixed eyes in 1% PFA then removed cornea and lens and put the eye cup (containing retina) in OCT for cryosectioning. The problem is that the protocol requires the retina to be fixed for short time and that makes it challenging to embed it in OCT as the retina is still soft. I am thinking to sections the whole eye without removing cornea and lens. Have you tried sectioning mouse eyes with lens on? Do you have any recommendation to avoid retinal breakage whole I remove the lens for OCT sectioning?

Thank you

Assraa

Dear all,

after 6 months of trials, we currently have a protocol that works for people who can not make eye cups without disturbing the retina.

We found the way of enucleating the eye is important. Cervical dislocation can cause retinal detachment, thus we use CO2 to euthanize the mouse.

When you scoop the eye out, make sure you do not pull the eye. It is the best if you still have a small chunk of optic nerve seen at the back of the eye when you cut it out.

Using an insulin syringe loaded with 4% PFA, we inject through cornea a small amount of fixative. Excess liquid will come out through the hole you punctured. Then we dip the eye ball in 4% PFA for overnight.

We use step dehydration of 10% 20% and 40% sucrose in PBS and finally mix 40% sucrose with OCT at 1:1 ratio and put the eye in that mixture for overnight before embedding with this mixture. This mixture will be softer to cut than OCT alone.

We cut the entire eye and see 3/4 eyes without retinal detachment.

But be aware that OS morphology is a little weird using this protocol. When we immunostain rhodopsin or PNA, we see a blob of distorted OS, instead of the nicely paralleled OS fibers as in the literature. If any of you know why this happens, please let us know.