Hi,
does anyone have any idea on how I can stain unmyelinated axons?
I am looking for a method to stain unmyelinated axons within a whole tissue, meaning that I will not cut it in sections but instead will be imaged as a whole.
Does anyone have suggestions on how unmyelinated axons can be stained?
I know BROPA method and DiOlistic but I think they just stain myelinated axons.
Thank you
Laura
Have you tried cytoskeletal stains like SMI-32?
Also depends on what kind of sections you are using - are these slices? Fixed or frozen? Cell culture preps?
No I did not try.
I will, thanks for suggestion.
Also as I said in the first post, I do not have section,the whole nerve will be imaged with x ray microtomography as a whole piece of tissue. No sections.
But I cannot find anything that stains unmyelinated axons.
Thanks
See attached paper for axonal staining in muscles. I have used this protocol to great success in diaphragms and other muscles.
Maybe you could try immunofluorescence:
One Neurofilament antibody that has worked well for me in cell cultures and whole muscle/ sections (for axon staining) is from Sigma. https://www.sigmaaldrich.com/catalog/product/mm/mab1621?lang=en®ion=US
I've attached two images (cell culture-- NF is shown in green and in muscle prep- NF is shown in cyan).
I use this antibody at a dilution of 1:250
In muscle sections, NF seems to work best with a shorter PFA fixation time. Not sure if these axons are unmyelinated or not but it works great!
Perhaps you could pair this antibody with one typical of myelin, such as Myelin basic protein?
Best of luck! I hope this helps.
Thank you all for suggestions.
However, I am still looking whole piece of tissue (no sections).
Do you guys think that would work on a whole tissue?
Thanks
IT should still work on whole tissues--- keep in mind antibody penetration will be more difficult in thicker samples.
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