I need the sciatic nerves unfixed for a subsequent assay. Before, I've embedded the nerves in 4% agarose and used a vibratome to cut 60 um sections. However, the entire nerve detaches from the agarose and becomes frayed by the blade during sectioning.
Is there a way I can cut these nerves without them falling apart?
hi Jane,
Fresh nerve not fixed !!! why?
if I have to do it I would do it this way: try the cryosection protocol without the fixation steps. and since the nerve is not PFA fixed, skip the graded sucrose embedding ( 10, 20, then 30%) and go immediately for the 30% but in a cold room, and embed with agarose little longer.
good luck
Dear Jane,
Do you have access to a McIlwain tissue chopper (not sure what department at CWU you are in)? It was developed by McIlwain to cut fresh hippocampal slices for electrophysiology, so it will cut thru fimbria-fornix. I have used it to cut fresh sections of goldfish brain (lots of myelinated tracts), since it is faster than using a vibratome, and in my hands does a better job. You might not even have to embed in agarose, but I am not sure about the size of your samples.
Good Luck!
Jill
Hi.
There are several tips in work with vibratome.
1. Agarose "base" should be more dense, than tissue. So, maybe, you should to use more concentreated agarose.
2. Nerves are covered by very tough membrane. Try to remove it before preparing of slices.
3. Use not liquid, but gel glue.
4. Some times it is helpful to make a groove in agarose's cube for tissue, that blade first cut agarose.
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