Dear colleagues,
I am planning to perform patch-clamp recordings using striatum slices from mice (young and adult).
I have been checking the methods from different papers and my impression is that the preparation of acute slices is done either by quickly removing the brain after isoflurane anesthesia, or after transcardiac perfusion.
In general, it looks that this is a delicate slice preparation and probably transcardiac perfusion is almost indispensable.
Does anyone with experience preparing these slices have a preference?
Is transcardiac perfusion a difficult procedure to learn for someone without this experience?
Thank you very much in advance for your contributions.
Best regards, Diego
Hi Diego,
I've done patch recordings in this area before, and In my experience, yes, the perfusion is needed.
In general, blood outside the vessels in the brain is bad news for tissue health. This holds up in sliced tissue as well. Bad tissue health makes for a bad patch clamping session. Plus, pre-perfusing before slicing can help you get the tissue temperature down before slicing and keep oxygenation up after the animal dies, again resulting in healthier tissue.
Specific to the striatum: The striatum has a lot of white matter, which makes visualization of cells more challenging, so anything you can do to reduce the amount of unnecessary material in your slice is great! A few free-floating red blood cells isnt terrible, but a whole lot can cause issues, especially if you have clot formation occurring.
The perfusion isnt a difficult process to learn at all. If you need any pointers on it, I'm happy to help!
Best,
Will
Thank you William D Marks !
I guess I will have no other option then :D
Just a couple of questions. Before the perfussion, would it be enough to anaesthesize the animal with isoflurane or is it preferable to use injectable anaesthetics, and in that case, which ones would you use?
I wonder if you might have a protocol that I could follow.
Thank you very much again for your help.
Best,
Diego
Hi Diego,
Our lab does trans cardiac perfusion routinely before cutting slices. It seems daunting if you never tried it. But with some practice it can be done routinely without much hardship. We anesthetize animals briefly with isoflurane before perfusion. As long as you open the chest and cut through the diaphragm quickly, animals will not wake up. Of course, this needs some practice. In the beginning, you can anesthetize animals more deeply with isoflurane, which will give you some extra time to cut open the chest.
Best,
Zhenbo
Dear Diego,
Both methods are equally efficient, but through trans cardiac perfusion you have to be very accurate and quick, which might be very complicated for someone with less experience.
So, I would probably recommend the acute slice perfusion, keeping the brain cool with cold acsf until is cut. Isoflurance is ok
All the best, Ioannis
Thanks a lot for the answers. I think I will give it a try with perfusion to optimize the preparation.
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