I am trying to optimize my antibodies for free floating IHC-IF on paraformaldehyde fixed mouse brain sections. I want to preserve my slices in an appropriate cryopreservation solution for later use and save their freshness. I can not order ethylene glycol due to some legal issues. They suggested diethylene glycol instead of ethylene glycol but I couldn't find any recipe or paper about its use on slice preservation. Is ıt possible to use diethylene glycol instead of ethylene glycol for slice cryopreservation? And lastly, can I use it with PVP40?
My protocol for cropreservation is:
Sucrose ----------------------------- 300 g
Polyvinyl-pyrrolidone (PVP-40) --- 10 g
0.1M PB ----------------------------- 500 ml
Ethylene glycol --------------------- 300 ml
Thank you in advance.
Hi Muhammed,
I can't comment on diethylene glycol but what I've done in the past is to cryopreserve the full brain in 30% sucrose in TBS before sectioning, then store the brain sections free floating in a 1:1 glycerol-TBS solution at -20C. I find this preserves them almost indefinitely for IHC with the exception of some particularly sensitive stains.
I agree with Nick Rose. With this cryopreservertion you can store your sections for years. That is my experience. If you do not work with TBS you can also use PBS to prepare your 30%sucrose solution. Very important: don't use this solution for cryprotection, because glycerol inhibits freezing!
Thank you for your suggestions. In my protocol, I perform transcardiac perfusion followed by 24 hours of postfixation with 4% PFA. Next, I put the tissue in 30% sucrose and wait for it to settle. I will try Nick Brose 's recipe, but I am also wondering, can I change ethylen glycol to glycerol?
Thank you in advance.
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