Hi @MGradwell. Thanks. Actually, I used another paper from your lab as the basis for a lot of my buffers this summer:
Overall it was pretty good and standard (sucrose cutting, and then regular aCSF).
Some specific points though about the slicing and lead-up.
- Do you do a full agarose embedding with melted agarose? Or do you make a cut-out from a solid block of agarose to hold the cord in place while slicing? And do you make any modifications to keep the cord from sliding out of the cut-out?
- Do you ever do transcardial perfusion with ice-cold aCSF or do you go right into the cord? I was using transcardial perfusion so that I could make the cord cold quickly, and that afforded me time to dissect the cord in the animal while still in the body. I know some people just cut out the whole vertebral column and put it in ice/slurry sucrose solution and then dissect in that, but I find it easier to take the vertebrae off when the cord is still in the intact animal.
- Up to what age animal do you use the sucrose cutting method? For animals older than 5 weeks, I know some have tried the NMDG recovery method (https://www.brainslicemethods.com/). Have you tried this or do you always use sucrose?
Thanks for your help.