Hi
I use patch clamp technique to do my PhD project. To prepare slides of brain samples, I treat them for one hour at 32 degrees Celsius in the cutting solution, and then I treat them for half an hour at room temperature. But I don't know, sometimes the quality of the tissue is not suitable and most of the neurons are depolarized. Can you guide me?
Hi Amin,
Welcome to patch clamp club! It's a battle to get healthy brain slices for Ephys. There are so many factors could affect the quality of your brain slices. Here, I will just mention a few of them. Hope this will help you. First, make sure your cutting solution and ACSF have correct PH and osmolarity. Second, I assume your cutting solution is ice-cold. Personally, I do not like too much ice in my cutting solution, which may change the osmolarity a lot. Third, I suggest you to cut your incubation time in cutting solution from one hour to 30-45 minutes. Lastly, you can try cardiac perfusion of animals with ice-cold cutting solution before taking the brain out. Depends the age of animals you plan to record. For young animals, mice for example, if animals are less than 1 month old, I do not think cardiac perfusion is necessary. But for adult mice (> 2-month), you will get better quality brain slices for recording if you do ice-cold cardiac perfusion.
Hi,
I have just started to work on brain slices too. I am not that good in it but what I found our by myself and other people suggested me:
1. Different brain reagons has different protocols. For example angel of tissue cutting so protocol between different brain areas can be slightly different.
2. Cell are very sensitive for different factors, but the strongest one is mechanical stimulus, or how other would say - how you handeling, transporting your brain slices. For example if you are saturating your cells' solution with too strong flow of carbogen gas it might generate mechanical waves that over time will damage your slices. Adding to that, if your slices are big then during transportation it can bend and damage cells.
3. Sometimes cell are depolarized not because of cell quality but problem in electrophysiology setup itself or how you approach the cell. What membrane R you see after patching? Cells that you patched that are around 1-2hr after your killed mice, if you keep cell at around -70mV for 2-3min, can you evoke action potentials?
4. Speed is very important. Cell are slowly dying, so shortening recovery period is better. On other hand, you will see better which cell are still good after longer incubation.
The other answers are spot on. I might add a couple other things.
* Anesthesia. If you use isoflurane (i.e. not perfusing), don't let it go for too long. Act quickly.
* Cutting solution. You can use regular ACSF, low-Na (high-sucrose), or NMDG-based cutting solutions. We prefer the high-sucrose version. We agree with Zhenbo about perfusing vs. age. After cutting, my lab incubates 20 min in regular ACSF.
* Cutting. Make sure your cutter blade is level (use Vibrocheck or equivalent), and the advancement speed is slow (we use 0.05 mm/s).
* When patching, use positive pressure to keep the pipette clean, but back off when you make your final approach to the cell, so it isn't injured.
* Random problems. This is electrophysiology, after all. For example, the mice might be sick. Sometimes, you just keep at it, and suddenly things are better. Good luck!
-Matthew
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