I am specifically working with human brain tissue. Most of the tissue has been in formalin for 2-10 years. I have read a couple papers claiming to have revived their tissue with certain heating+buffers protocols. I have tried a few protocols and have gotten very poor protein quantities in my western blots.
Any suggestions or information about formalin-fixed tissue would be very helpful!
There are multiple issues with that.
First, formalin cross-links the proteins in the tissue. If the cross-linking was done correctly, in theory you should have proteins cross-linked with whatever molecules happened to be next to them during fixation. This changes their molecular weight in unpredictable and heterogenous ways. So you should expect a lot of heterogeneity and smearing. You can hope to find some un-modified protein running at its native molecular weight, especially when the fixation protocol was executed poorly (old formaldehyde, contaminants...). If you can believe it, people virtually never use ideal conditions for this. And that's of course the big variable that isn't reported in these "revive my fixed proteins" papers...
Second, the cross-linking causes chemical changes to the epitope that's recognized by your antibody. This can call for higher antibody concentration, weaken your signal, or abolish your signal. When you purchase your antibody, it's important to check if it has been tested with fixed material. Some antibodies work only on natural proteins. Some work on both natural and fixed proteins. There are even some that work best on cross-linked proteins. If it doesn't say it online, you can call your antibody vendor and ask about it.
Good luck, hope it helps...
There are multiple issues with that.
First, formalin cross-links the proteins in the tissue. If the cross-linking was done correctly, in theory you should have proteins cross-linked with whatever molecules happened to be next to them during fixation. This changes their molecular weight in unpredictable and heterogenous ways. So you should expect a lot of heterogeneity and smearing. You can hope to find some un-modified protein running at its native molecular weight, especially when the fixation protocol was executed poorly (old formaldehyde, contaminants...). If you can believe it, people virtually never use ideal conditions for this. And that's of course the big variable that isn't reported in these "revive my fixed proteins" papers...
Second, the cross-linking causes chemical changes to the epitope that's recognized by your antibody. This can call for higher antibody concentration, weaken your signal, or abolish your signal. When you purchase your antibody, it's important to check if it has been tested with fixed material. Some antibodies work only on natural proteins. Some work on both natural and fixed proteins. There are even some that work best on cross-linked proteins. If it doesn't say it online, you can call your antibody vendor and ask about it.
Good luck, hope it helps...
FA fixation is reversible. The issue is how to minimalize sample loss first. I would suggest to use ammonia vapour (i am a chemistry major) and some heating on the rehydrated sample, than extract it That is a suggestion, not a worked-out method.
Unfortunately, it will be really hard. It depends heavily on the way how the tissue was stored and how long has it been stored. Since I work with human brain tissue, I know how hard it is to get it, but I suggest you try (not really you but your PI) to find fresh or frozen tissue.
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