I doubt the quality of my slices. Neurons in the surface layer completely die (which is quiety different from P17-21 pups, sometimes has nice alive cells in this layer), and have to penetrate to deeper layer for patching, during which my pipette seems be sealed by debris. Anyone would like help me out by sharing your suggestions? Thanks in advance!
Thanks, Mehdi. I used 206 mM sucrose solution with high Mg and zero Ca. I hate this project but no other option and have to deal with mice with this age. I need optimize the condition to make it work effectively
Try higher resistance electrodes (8-10 Mohm) and avoid lateral movenent once you are in the slice
Hi, I have never tried it, but volks are speaking about an option which can improve quality of slice even from older mice. After anesthesia, try to perfuse the mice transcardially with the ice-cold and bubbled (oxygenised) cutting solution (in your case the sucrose -.high Mg - zero Ca fluid), Then you can remove the brain from the head of the mouse and cut slices with the vibratome in the same solution. In this case the ice-cold and oxygen-saturated cutting fluid can reach the neurons of the CA region much earlier then in any other case, so damage is less. Good luck!
Thanks, Imre. I hve tried and found the slice quality significantly promoted. I'm still trying minimizing lateral movement as Prof. O'Donnell's suggested. seeking the good luck come soon
Thanks much. I have talked with the company and I'm going ahead to try it out!
Yongfu,
Perhaps your vibratome needs to be recalibrated. That is what happened to mine. I was about to buy a compresstome when I decided to try to have my vibratome checked out. Something was wrong with it (Vertical axis motion).
I have had better luck and slices since.
Thanks much, Olivier. I'm going to check the VT1000s to see what could be wrong. One of professor around this city informed me that the VF300 did give him better surface neurons, but the machine has lot flaws need to be serviced, which costs much $$$
Hi Yongfu, I am the author of the SFN meeting posters (one in 2011 and one in 2012) mentioned by Tomasz Wójtowicz. I am able to record routine whole-cell patch from CA1 pyramidal neurons in acute brain slices from adult and aging transgenic mice with very high success. I recently recorded CA1 pyramidal neurons in whole cell mode in slices from a 2.5 year old transgenic mouse with excellent success. I have developed improved methods that enable this type of patch recording and shared many details of the protocol and solution recipes on the following website
With these methods you will be able to see healthy cells very near to the surface of the slices (within 20um of the cut surface). For CA1 you should pay special attention to the glutathione restoration strategies that I describe, as this is the most helpful trick to ensure very good preservation of the pyramidal cell layer in adult brain slices. It is also important to ensure a proper slicing angle that produces essentially transverse hippocampal slices in which the CA1 payer is relatively tight. If the layer fans out the angle is not optimal and this will make things more challenging. The Compresstome is helpful for adult brain slice but the main trick is the recipes and the procedures. I hope this helps you out.
Thank you very much for your kindness, Dr. Ting! I have searched all yours or your volks abstracts/publications , and trying follow the protocols. However, I still have problems. Can you share me your email address/lab phone that I can turn for help?
Thanks in advance.
My email is [email protected].
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