Hi,
I have brains that express GFP under a promoter of an endogenous protein so they actually express a fusion protein.
I want to visualize GFP and thus know where the endogenous protein expresses (i.e. in what brain regions).
Do you think I can post-fix the mice or a perfusion is needed?
Is it better to visualize GFP in floating sections or by staining for antibody against it?
For antibody staining, which one, DAB or IF is better? I am afraid that blood vessels will have endogenous fluorescence especially if I do not perfuse so they will be false-positive.
What do you think?
Dear Iddo,
I have always perfused my animals that express GFP, but that is what I need to do in order to use the same sections to localize other proteins. Personally, I don't think that immersion fixation of brain tissue gives very good protein preservation unless you are able to dissect out the specific area of interest, especially if from adults.
Depending on the strength of the promotor and your imaging system, you may need to use an antibody to enhance the signal.
Please realize that if you do not perfuse the tissue and use DAB, you will still have some background staining in the red blood cells in the vasculature. It's sometimes difficult to use enough H2O2 to block their reactivity to the DAB.
For a quick initial survey of the brain, you might be able to quickly image freshly dissected brains that have been sliced in a brain matrix, and then kept in oxygenated ACSF (sort of like brain slices for electrophysiology). That would give you an idea of signal strength, and what you might need to do next.
Good Luck!
Jill
Hi Jill,
Thanks for replying.
i did not understand the comment: "Personally, I don't think that immersion fixation of brain tissue gives very good protein preservation unless you are able to dissect out the specific area of interest, especially if from adults"
Waht do you mean by "dissect out the specific area of interest"? What if I can dissect it out?
Iddo
Jill,
What if I all I need to know is absence/presence of GFP, not quantitative staining? Does it change the method of choice?
Iddo
Dear Iddo,
Aldehyde-based fixatives can only penetrate at a certain rate into tissue. So if you have a small piece of tissue you can immersion-fix it. A slice made from the brain is probably OK to immersion fix. There are fixatives that penetrate more rapidly, but they may not be compatible with looking at your protein of interest.
Brain tissue deprived of oxygenation can degenerate quickly. This is one of the reasons that people perfuse the brain-so that they are sure that the neurons are as close to representing their in vivo state as possible when they are later processed. It is always a trade off--you don't want to over-fix the tissue, but the histology and protein localization needs to be preserved. With immersion fixation of thick tissues, because of the limited speed of penetration, you can end up with overfixed outer layers of tissue, yet underfixed inner layers. You might want to look at CLARITY and other similar methods to see how they have fixed their tissue. I realize that you may not want to perform multi-photon imaging of an entire brain, but it will give you an idea of good ways to maintain GFP localization.
If you want to look at living tissue, but don't have access to a brain matrix for making slices, you could also use a vibratome, or a McIlwain tissue chopper.
Alternately, you can freeze the unfixed tissue in a quick but controlled manner, and then cryostat section the material. Since the cryostat sections will be thin and the tissue is not fixed, I would suggest collecting the sections onto slides and then quickly fixing them. But in this way, you could limit the exposure of the tissue to fixative, if that is your concern.
Unfortunately, there is not a "one size fits all" solution. For faint GFP expression, you might be better off fixing the material and amplifying the signal with an antibody to GFP. You will just need to try a few different approaches.
Good Luck!
Jill
Jill,
Is one hemisphere of mouse brain small enough for PFA to penetrate?
Also, do you think there is any advantage of using frozen sections/paraffin sections?
After post-fixation, do I have to switch to a lower PFA concentration prior to cutting?
Thanks
Iddo
Dear Iddo,
Hopefully you will get more input from others here on RG regarding your questions, since the largest piece of tissue that I have ever immersion-fixed is an adult zebrafish brain. Even then I was a little hesitant to do it, but all I needed to quickly look at GFP expression in order to see where to perform the in vivo imaging. A zebrafish brain is pretty small-maybe the size of the two mouse olfactory bulbs?
I do not use paraffin sections for my work. It is much easier for me to use cryostat sections. The trade-off between the two is that paraffin sections result in excellent histology, but at the cost of some proteins (and certainly lipids). If you are thinking of using paraffin sections, your tissue must be well fixed before embedding. In order to visualize GFP in paraffin sections, you may want to try these procedures for fixation and embedding:
https://www.ncbi.nlm.nih.gov/pubmed/26345508
https://www.ncbi.nlm.nih.gov/pubmed/20147043
I do not understand your last question: "After post-fixation, do I have to switch to a lower PFA concentration prior to cutting?" I think would depend on the technique that you are using to section your material. I always fix with one concentration of PFA, since I use the perfusion method. If I am cutting cryostat sections of fixed material, it must be cryoprotected in a sucrose solution to avoid freezing artifacts. I do not add paraformaldehyde to my sucrose solutions. However, I know some people who use a freezing microtome for cutting thicker frozen sections who do.
The only time I have stored material in a low concentration of paraformaldehyde prior to sectioning is when I was attempting to perform DiI tract tracing in fixed material. Since it takes a while for the DiI to diffuse down the lipid bilayer of the axons, you need to stabilize the tissue.
Sorry to be so unhelpful.
Regards,
Jill
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