you can try using a chilled Choline Cloride solution (see attachment) instead of NMDG.

Old animals are difficult anyway. perfuse with choline chloride, cut in choline chloride (max whole time in the solution 15-20 min), the incubate at 32 in ACSF and then at RT. Cut slices of 250 uM. Good luck !

Hi, Guangling!

Does your cutting solution contain substances reducing edema and oxidative stress in slices? Aged neurons are vulnerable to these factors. Here is a good paper explaining what happens with brain slices during cutting procedure:

Article Acute Brain Slice Methods for Adult and Aging Animals: Appli...

Also, it is important to do cutting without delay and after that to maintain temperature during slice storing.

I hope this will help you,

Oleksii

Thank you for Oleksii 's advice ,I have read the paper you recommended before.My recipe of NMDG come from this parper also.But I will read it again to find some details, may be key to solve my trouble.Thanks Paula! I never use Choline Cloride solution before,can you tell me the max whole time in the solution contain perfuse and cutting stage?Now,I use NMDG -ACSF perfusing and cutting,then transfed into HEPES -holding buffer for incubating, the slices can be recorded in spite of not very easy job.

the max time in Choline Chloride is 20 min, 20 min is enough to perfuse and cut the slices. Then you transfer them to a normal oxygenated ACSF. We use 32 C for 1 hour and then switch to Room Temp, to prevent fast degardation of the slices. you don't need HEPES if you are oxygenating your solution. if your problem is sealing you might consider changing your internal solution. and set your osmolarity around 300 mosm for external and 290 for internal.