Anyone ever make coronal sections of spinal cord so that you can see multiple segments in one view? How thick? Cryostat Or vibratome?
Like this one:
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Amaury says he has done 50um on a cryostat
Thanks! I’ll give it a try.
Yeah I do this a lot to visualize projection neurons since the you can see the dendrites and cell morphology more clearly in this plane (I have also seen it called the horizontal plane since “coronal” can be confusing in parts of the body that aren’t the head). I do them the same way I do all my spinal cord sections, which is cryostat with either 14-20um for slide-mounted sections or 40um for free-floating immuno applications.
Excuse the terrible image quality, but this is what my sections look like through lamina I-II where you can see brightly CTB-labeled PNs.
That’s great @liz! Thanks.
So, I’m assuming you just mount the cord sideways and slice like that?
Do you start from the ventral face first and work your way to the dorsal, or start with dorsal?
When I made the sections for the image you’ve linked above, I did it at 40 um thickness, with the spinal cord placed horizontally in OCT as you have in your sketch. I tried cutting dorsal first and ventral first, and didn’t see a meaningful difference either way. In practice, what matters most for getting this sort of image is how flat you can get the cord to lie in OCT, and how much the cryo is behaving on that day.
Some tricks I used for this image: I pre-froze half of my mould with OCT, and then applied a very thin film of OCT that I immediately then placed the cord onto. This thin film would harden very quickly from residual coolness from the frozen layer, but remain flexible for long enough to allow me to straighten out the cord and ensure it lay flat. Once the thin layer had frozen in place, I then added in the rest of the OCT to fully enclose the cord.
These were directly mounted onto slides, not free-floating.
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Thank you @shrivatsiyer for joining this forum and for sharing your tips on how you made such beautiful spinal cord slices. This will come in handy. I’ll try it your way and report back.
I mount mine with the dorsal side up so that I can ensure that the cord is straight and flat, and because when I use this plane of sectioning I’m almost always looking at lamina I. I think my approach is very similar to @shrivatsiyer - I create a flat surface by dropping some tissue freezing media (TFM) onto the cryostat chuck and then placing a heavy flat weight on top of it. Once I have a flat surface, I make a strip of TFM that I immediately placed the cord into. While the TFM is still transparent I can roll or adjust the cord to make sure it’s flat, but there’s only a very short window before the TFM and cord will start to harden.
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