Keeping a spinal cord slice anchored/fixed in a perfusion chamber for calcium imaging

Experimental Techniques Protocols
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1

Does anyone have a good solution for keeping a spinal cord slice (400 um) anchored in place in a perfusion chamber? When I was doing calcium imaging the other day with a slice, throughout the experiment the slice would move ever so slightly, presumably due tot he force of the perfusate. This messed up my imaging since the ROIs borders move off of the cells.

I was using a weighted U-shaped bar with a thin wire mesh band to hold the slice, which I understand is what people typically use for electrophysiology. Is there anything more robust though? What about coating a coverslip with poly-D-Lysine or something? Will that work?

@fmoehring

2

Sorry we don’t usually perform calcium imaging on spinal cord slices. And to anchor things we have also only used a weighted U-shaped bar. Have you tried fixing the U-shaped bar in place with some pins?

3

Thanks @fmoehring I’ll try using pins to anchor the u-shaped bar. That might provide more stability.

4

Looks good but the perfusion causes the ROIs to shift. Gotta fix that.

5

Alternatively, can you use landmarks to align the frames such that the ROIs are aligned during analysis?

6

Hello everyone! Here’s another common question we receive at Precisionary Instruments:

How do I hold down slices in my recording/imaging chamber?

Although we sell Compresstomes and other tissue slicer equipment, we have a whole team of scientists and engineers to help our customers with these types of issues. The vast majority of our customers are researchers.

Here is the quick answer:

Harp: Use a stainless steel “harp” to hold down acute spinal cord slices for electrophysiology and imaging experiments. We have had customers recommend the following place to order harps in all sorts of shapes and sizes:
https://www.warneronline.com/product_info.cfm?id=80

Poly-lysine coverslips: Not many people know about this research “trick” for helping slices stay immobile during experiments. Take some miniature round coverslips, such as these:
fishersci.com

Microscope Cover Glasses: Circles - Microscopes, Slides and…

Packed for easy dispensing; clean and ready-to-use.
…Soak the coverslips overnight in 10% ethanol (this helps clean the surfaces so that tissue will adhere better later on). Then spread out coverslips on a paper towel or large petri dish. Pipette poly-lysine solution onto each coverslip. Make sure that the surface tension holds on each coverslip, such that the solution doesn’t “break” and spill everywhere. The poly-lysine solution we use is:

http://www.sigmaaldrich.com/technical-documents/articles/biofiles/poly-lysine.html

…Incubate the coverslips for a few hours, until the poly-lysine solution has evaporated. There will be a thin layer of poly-lysine left on each coverslip surface. when you put a spinal cord tissue slice on it, use a kimwipe and soak up the extra solution so that the tissue touches the top surface of the coverslip.

You can now place your slice-on-coverslip into your recording chamber and do your experiments! The HUGE advantage of this poly-lysine coverslip method is that there is no damage to the surface of the tissue slice from a harp. The entire surface is smooth and free for patch-clamping. In addition, the coverslip will also hold the tissue and make it immobile for experiments like calcium imaging.

We hope that this helps! Please let us know if you have any questions!

Thanks,
Compresstome Team
info@precisionary.com
www.precisionary.com