Hello everyone
I recently started working on a project concerning the RPE and the retina.
I want to perform IHC on paraffin embedded eyes, but I have problems to find the right fixative because the RPE detaches really quick from the 'neural' retina.
I now found that Davidson's fixative is the best one to preserve the structure of the retina, but it seems that it is not compatible with IHC.
I used the following recipe for the Davidson's fixative: (per 100mL)
- 22.2mL 37-40% formaldehyde
-33.3mL ethanol
-11.1mL glacial acetic acid
-33.3mL destilled water
Did I use the wrong recipe or does anyone have a possible solution for my problem?
Thank you in advance
Hi Yannick,
For most antigens, fixation of mouse eyes using 4% paraformaldehyde in 0.1 M PBS or HBSS buffer at pH 7.2-7.4 for 2 hours to overnight will work very well and can provide excellenrt preservation of retinal tissue. My lab uses this as our standard fixative as do many other retina labs. (Cacodylate buffer will work, too, if that is your lab's preferred buffer). For more sensitive antigens shorter fixation may be essential to preserve antigenicity. My own experience with Davidson's fixative for immunofluorescence has not been good. You also may find that paraffin embedding denatures many of your antigens to the point where you may not be able to immunolabel them. There are antigen retrieval methods to help deal with this problem, but they don't always work for all antigens. Frozen sections are often a better bet for immunolabeling, but they have their own challenges.
As suggested above, if you can perfusion fix the mouse, it may lead to better tissue integrity, but immersion fixation of eyeballs works well, too. A key thing in preparing mouse eyeballs is that you must handle them gently because, as you have experienced, the neural retina detaches from the underlying RPE pretty easily. If you are working with a mouse strain that has a retinal degeneration the tendency to detach gets worse due to the pathology.
I have attached a fixation and preparation protocol that my lab uses for preparing mouse eyes for frozen sections. The preparation would be identical for preparing eyes for paraffin embedding (if that is a critical need for you) through the buffer rinses to get rid of the excess fixative. After that step, you would then need to go through the dehydration steps (with alcohols usually) needed to embed the tissue in paraffin.
I have had the pleasure of visiting Leuven a couple of times. Its a lovely place. I hope all is well.
Best regards,
Dave Sherry
Hi Yannick,
I guess you are not talking about staining on flat mount retina, right ?
Because if you are just cutting slices, I would say that when you are dissecting the eye, you should just take the cornea out above the ora serrata, and then being very gentle with the tissue should be enough for you to keep retina and RPE altogether, whatever the fixative protocole 🤔
But, if you're trying to flatmount your retinal tissue with the underlying RPE, then it's a little bit random, it depends on how carefully you behave with you samples ...
Best,
Hugo
Yannick
First what animal model are you using? some retinas are more prone to separate than others. Second, the acetic acid in your fix (11%) is probably disrupting your ability to do IHC because it denatures/precipitates proteins. All you need to do is to isolate the retina from the globe by removing the cornea as Hugo stated above, and use a buffered 3.7% formaldehyde solution. Best to do this at room temperature for 30-60 min since it allows the fixative to permeate into the tissue faster than at 4 degrees C.
Good fixation of the retina:
please read the attached files.
Dear Yannick,
The mouse retina has a very thick inner limiting membrane covered with blood vessels and does not easily seperate from the retina. I recommend that you should perfuse the whole animal with intracardiac perfusion method before getting the eyes out. If you cant use that method, your second best option would be to get the both eye balls out after overdosing your animal with anesthetics. Then make incision with fine scalpel tip on both sides of the eye balls to let fixative into the tissues. I use 4% PFA in PBS (see below), and leave eyes in the fixative overnight at 4'C- Then wash in 0.1 M PBS 3 times to remove fixative. Then incubate the eyes in sucrose solutions to prepare for freezing. Mount on cryostat metal holder at -20'C and cut sections (~20 - 50 micron) thickness. This should give nice big sections of the entire eye including the retina and RPE attached together. I usually prefer floating sections for immuno, though you will have to be very gentle with your sections throught the entire processing.
best wishes,
Refik Kanjhan
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