This is a long-shot question. I have some methanol-fixed tissue neural tissue. I'm wondering if it would be possible to get intact single cells from the tissue once rehydrated. I was thinking mechanical dissociation with trituration. If this were fresh tissue, I would use a protease like papain first to help the dissociation, but since this tissue is methanol fixed, I'm concerned that (1) the protease won't work (2) it may work but it will go intercellularly and mess up the internal proteins.
Is there any precedent for dissociation of fixed tissue?
Hi, Alex.
Forget about the isolation of single cells from the methanol-fixed tissue. Not possible, sorry.
However, if you aim protein analysis of the fixed neural tissue, then gradual rehydration giving enough time at each steps (~15-20min). to release remaining methanol and non-methanolic contaminants contained in it, i.e., 95%, 90%, 80%, 70%, 50%, 30%, 10% methanol, ddH2O(I), and ddH2O(II).
Then snap freezing the rehydrized tissue in LN2 gas. Transfer the frozen tissue immediately on pestle and motor, grind the tissue while adding LN2 gas. Extract proteins from the tissue powder. This will contain whole neural tissue proteins, including cellular proteins from neurons, glia and their ECM.
This is the only option unless you prepare fresh tissue again.
Good luck!
What would you like to do with the isolated cells? Have you considered Laser Microdissection? Maybe this would help, and you can easily use it for fixed tissue:
http://www.molecular-machines.com/single_cell_sorting_products/_mmi_cellcut_-_laser_microdissection/technology_27
Hi,
Just seeing this thread now, but I am going to answer this here in case it is helpful to anyone who might stumble upon this .... You could look up this website: www.rnassist.com. This product called vivoPHIX enables tissue fixation and then dissociation for tissues, prior to scRNA-seq.
Sarah Hélène Merkling - vivoPHIX sounds really interesting, have you used it yourself to process tissues + dissociate for scRNAseq? Would you mind sharing your experience with it? Thank you!
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