Hi scientific community!
I was hoping to get some input on a problem I am currently having deciding how to fix my samples for AFM. I am using AFM to detect stiffness of the ECM in fibrotic areas, and have the option to leave my samples unfixed (we have tested that this works), or go for PFA, NBF or a solvent method like methanol.
Since we know methanol will dehydrate the ECM and PFA or NBF will cross-link it, we were intending to go straight for measuring the unfixed tissue, since this would be most "correct". However, whilst the main structural proteins like collagen will be fine for the time period of the AFM experiment if kept cool, it has come to my attention that other smaller, less stable ECM proteins could degrade if unfixed, which could affect the integrity of the fibrosis and therefore the resulting stiffness measurement.
Most previous literature either uses unfixed tissue or PFA.
What seems the best method to you - to fix the ECM, or to go ahead unfixed?
Why not do the experiment? Measure a fresh tissue, fix it (PFA), measure again.
But to answer your question: The closer you are to the living state the better, scanning in buffer would be the best. The ECM is fairly robust in comparison to other structures, I would assume that unfixed will give you the most accurate stiffness measurements. Once you aldehyde-fix biology a lot of unpredictable things happen (tissues turn brittle, proteins are crosslinked but nothing else), and solvents are even worse because a lot of material gets extracted. Also keep in mind that NBF contains methanol. And all these parameters vary depending on which tissue you're looking at. You'll have to do the experiment! ;)
For general structural preservation PFA>NBF>solvents
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