Hello,
I have a problem with the cryoprotection of mouse hippocampal slices. The samples are PFA-fixated and cryoprotected in 30 % sucrose in PBS solution; they would normally sink to the bottom of their wells in the 96-well plate after a couple of days in the cryoprotectant, but this time none of the samples (there are five of them all in all) would sink. I have already encountered such a problem a couple of times before, and according to my experience it is not a good idea to proceed with samples that are still floating, as that implies that they have not been cryoprotected well enough and therefore are very likely to get damaged during sectioning.
I placed them on a shaker in a cold room now, as I hope that that would help the cryoprotectant to enter the samples; however, I am not sure that that would work. Therefore, any advice would be very appreciated.
Thanks a lot in advance!
Max
Hello,
for a whole mouse brain I use 100 ml of your cryoprotective solution.
Why don't you incubatue your slices in a 24well plate with 1 ml solution?
Good luck
Stefan
Dear Hussein,
I suppose that Maksims is working with slice cultures in vitro.
Perfusion is not possible then.
Sincerely Yours
Stefan
Hello Stefan,
Thanks a lot for your advice: it sounds like a really good idea. I used to use a 96-well plate rather than a 24-well plate, as it would work fine in nearly all cases: I had the problem with floating slices just a couple of times out of several scores of slices I have processed so far. However, I think I should switch to using a 24-well plate to make cryoprotection more reliable and perhaps faster as well.
So, I have just transferred the slices I'm currently cryoprotecting from a 96-well plate into a 24-well plate. In addition to that, I am trying to fix the problem with these particular slices by using 15% (rather than 30%) sucrose in PBS now; as soon as the slices will have sunk in 15% solution, I will exchange it for 30% solution again and keep them in it for a day more.
Thank you once again! I'll post some information about the outcome of this experiment asap.
Best,
Max
Hello Hussein,
Thanks for your advice! However, as Stefan has rightly observed, I do work with slice culture in vitro rather than go myself through the steps that you have described, so I am mainly concerned with cryoprotection, cryotomy, and staining rather than with what precedes that.
Best,
Max
Hy again,
I forgot to mention that I use meanwhile 20% sucrose and that is working well.
Sincerely Yours
Stefan
Hi Maksims, to run a sucrose gradient raw is a good idea. You can also start with 10%, 20% to 30% or smaller steps like 10%, 15%, 20%, 25%, 30% and stoer in 30% over night. Use bigger vials like 1,5 ml or 2ml Eppis. I am working with slice cultures as well as with 300-400µm thick brain slices after electrophysiiological investigations. We put the sections after fixation in this cups for a couple of days and get good results. To control the quality I recommand a NIssl-stain with cresylviolet. Good luck.
Hello Ute,
Thanks a lot for your advice!
After having tried to run a sucrose gradient with my samples recently, I think it's the way to go indeed. Incidentally, I had been wondering for how long time you keep samples during each of the steps: for instance, do you keep them overnight in 10%, then overnight in 20%, and then finally overnight in 30% sucrose, or do you use the steps of some other duration?
Thanks a lot in advance!
Best,
Max
The duration of each sucrose concentration depends on the sice of the tissue block. Change the sucorse solution after the blocks has been sunken on the bottom of the vial.
Hello Maksims,
I do have the same problem with neurospheres. Sometimes they sink and sometimes not.
Did you solve your problem?
Best Julia
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