I’ve been unhappy with my slice quality lately and I’m looking to improve slice health. I’ve tried various times/variations of simple extractions and also transcardial perfusions. I just can’t seem to find the right combination. I’ve tried one to three minutes of perfusion with carbogen bubbled acsf without fantastic results (I clamp the abdominal aorta). Also, I was wondering if anyone had success using HEPES in a holding solution rather than just holding the slices in recording ACSF. (I’m doing recordings on 90-120 day old rats, by the way.) Any input would be really appreciated!
you can try a choline chloride solution instead of ACSF. Clamp the aorta and perfuse ice cold solution until the eyes are clear and nose is less pink. Use the same solution to slice. Total time perf +slicing should not be more than 20 min.
Paula Parra I actually do use a choline-substituted "cutting" ACSF to perfuse and slice. About how long would you say that you end up perfusing? Thanks so much!
For older mice (12+ weeks), we've had good results using an NMDG-based slicing and recovery procedure.
Article Preparation of Acute Brain Slices Using an Optimized N-Methy...
Unlike in the JoVE video, we slice with a vibratome and so the slicing takes longer, but otherwise our procedure is the same.
At the Allen Institute, they use this for human tissue, and so there's no reason to think that it shouldn't work with rats too.
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