cryoprotection mouse brain sections

[martikacska]

Hi,

Does anyone have any experience with storing mouse brain sections on -20 in ethylen glycol glycerol? Is it better than on 4 degrees in PBS+azide?

Thanks.

[excalibur]

Are they fixed in formalin?
How long are you planning to store them?

[CanuckPhD]

Our lab has stored thick (30um) sections in PBS/azide for a few months in the fridge without any issues with our staining (microglia, astrocytes and T cells). It is important to make sure the tissue is well fixed though before you do this (48 hours in 4% PFA works well for a rat/mouse brain).

I haven't used the -20 and glycerol but have heard that it works fine.

Good luck

[martikacska]

Hi,
The brains were immersion fixed in 4% PFA for three days, equilibrated in sucrose, cryosectioned...I would like to keep them for few months...I was having bad experiences with PBS/azide, that's why I would like to switch to glycerol and -20. I heard from someone that azide does not block proteases which are in the tissue and do work to certain extent after fixation as well. This is why I thought -20 is safer. What are your experiences?

Thanks

[bjchoi]

I store much of my tissue in a similar cryoprotectant solution.  We use 30% ethylene glycol/20% glycerol/50% formalin or PBS.  We've kept tissue stored like that for years.

Hope that helps...

[jfrasca]

We use a mix of 30% glucose, 30% ethylene glycol and PB as cryprotectant and we store it in the -20 without any problems. We just add an extra wash or 2 when we start IHC to make sure we get it off. 

[richard03]

Here are a few protocols form cryoprotectants.

http://www.ihcworld.com/_protocols/histology/cryoprotectant.htm