If you use spinal cord slices, would you mind describing your procedure for cutting them?
In our lab, most people cut out the cord, and then use a block of agarose (4%) with a small half-cylinder cut out to support the cord. Then it is glued and set perpendicular to the blade on a Leica 1200VT vibratome. 300-400 um sections are then made. This works well enough but sometimes the cord pops out of the hole. Some people remove the dura, some don’t. But the ability to make the slices in this way seems to depend on the quality of this vibratome, which can cut sharply and not smash the tissue.
Some protocols I’ve seen do a full agarose embedding. This definitely helps keep the cord stable during slicing, but the fact that the cord is not surrounded by cutting solution, but only the agarose isn’t optimal for slice health.
I’m trying to use an older Leica 1000VT this summer and using the standard method from my home lab, I mostly am smashing the tissue and not making cuts.
we have another machine called the Compresstome (http://precisionary.com/) that has a different mechanism requiring full embedding. I’ve done this and macroscopically the slices are good, but the other electrophysiologists here have said the health of the slices are poor. I have yet to patch with them.
Qing-song Liu, Ph.D. from the Department of Pharmacology and Toxicology at the Medical College of Wisconsin pointed me to these tips for cutting tissue including spinal cord (see links below). He stressed replacing sucrose with NMDG-Cl during slice cutting, transcardial perfusion of NMDG-based cutting solution before taking the spinal cord out and embedding the spinal cord in low-melting point agarose (this last one we were already doing).
Thanks @RPSeal. Does your lab do the full NMDG method or use sucrose? It appears to depend on the age of the mice. They recommend for 5 weeks or older.
So far just sucrose but we have some new scientists in the lab so may venture into seeing if these methods improve our recordings. We typically use mice that are 3-4 weeks old because we are recording in lamina III.
I’m trying both here this summer. For the younger animals I’m going to stick with sucrose. But for anything 5 weeks and older, I’ll use the NMDG. I’m doing full embedding in low-gel agarose, but I find it difficult to get the agarose off the SC without tearing. I’ll have to play around with the percentages.
Hello! This is a great discussion about cutting live slices for spinal cord electrophysiology. I wanted to respond on behalf our company, Precisionary Instruments, which created and makes the Compresstome vibratome.
We originally designed the Compresstome for cutting live slices for researchers in neuroscience. The Compresstome has these advantages:
Faster cutting speed: Compared to the Leica vibratome models, the cutting speed is faster, but the slices are just as healthy. The time saved for cutting helps prevent neurons from dying, especially when you can get the slices from the cutting solution to the incubation chamber in a shorter amount of time.
Decreased deflection: What does this mean? When you’re cutting with a vibrating microtome, you want the vibration to move left-and-right, not up-and-down. Deflections in the up-and-down direction causes shearing of the top surface of acute slices. This means that the surface cells will be damaged and will not be viable for e-phys. The Compresstome models with Auto-Zero Z technology (VF-200-0Z, VF-300-0Z) have a vibrating head that has been pre-calibrated for cutting where there is <1 um z-axis deflection. This yields healthier slice.s
Embedding for stability: The Compresstome requires that tissue specimens be embedded in agarose. We use low-melting temperature agarose so that the tissue’s temperature does not get too high. Embedding in 1.8 to 2.5 % agarose allows the entire tissue sample to be stabilized. For spinal cord tissues, you can place a the spinal cord onto a long piece of agar or solidified agarose, then insert it into the Compresstome specimen tube. Embed the contents of the tube in agarose, and the entire specimen will be stabilized for cutting.
I hope this helps answer some of the running questions about what vibratome to use for cutting spinal cords! We take great pride in helping our customers, so please don’t hesitate to reach out to us!