I am dissecting single muscle fibers from previously frozen tissues (flash frozen in liquid nitrogen and stored in -80 for a few years). After placing in skinning solution as reported by Trappe and the Ball State research group since 1985, the fibers were lysed as expected. Upon transferring into relaxing solution also as reported by this group my muscle sample is falling apart into tiny fragments and the fibers become VERY brittle and break under minimal tension. I have heard of others using RNAlater for previously frozen tissues, is this the best option? Can I change the relaxing solution at all to prevent this from happening? Or, is this due to old frozen tissue that may have been through a few freeze/thaw cycles and compromised the cell membranes to begin with?
Snap freezing in LN2 results in muscle fiber damage due to ice crystal formation. It is suggested to store tissue samples in various combinations of glycerol/relaxing solution.
https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1175&context=kaleidoscope
Thanks, so flash frozen tissues in LN2 are less than ideal for fiber isolations...I am trying to use some old stored tissues to pilot a method. Might be better suited to just use fresh from your response?
Yeah, unfortunately its unlikely you'll be able to isolate quality single fibers - fresh samples would definitely be ideal in your situation. Good luck!
So we started isolating fibers in our 50% glycerol skinning solution and it has solved the issue. Not too sure why the relaxing solution isn't working as designed but thanks for the input.
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