[Histonet] Re: Glycerol cryoprotected tissue and cryostat

[Delatour Benoît]

>The protocol said to clear
>in glycerol which we did and the tissue is in 80%
>glycerol.  I have spent the morning trying to cut
>this tissue on a cryostat without success.  When
>I put the mouse placenta in OCT using isopentane
>and liquid nitrogen, the OCT freezes but the
>tissue does not.  Cutting it on the cryostat is
>then not possible.

How long does the tissue remains in glycerol? Actually glycerol is a very 
efficient cryoprotectant. Usually you use it at lower concentrations 
(10-20%) associated with dimethylsulfoxide (DMSO)to potentiate tissue 
penetration. 80% is very high! In anycase placing overnight tissue in a 
2%DMSO - 20% glycerol solution prevents cutting the specimen with a 
cryostat because the temperature is too high (around -20°C) and prevents 
freezing of the tissue. On the other side this cryoprection method is 
perfect for sliding microtomy (because you can reach lowest temperature 
with such apparatus).

What I would recommend (if compatible with your IHC protocol):
-remove all glycerol by repeated baths in PBS
-re-infiltrate tissue with a less potent cryoprotectant (eg sucrose) till 
equilibrium
-cut with cryostat

Benoit

--------"If you think research is expensive, try disease"--------

B. Delatour
Laboratoire de Neurobiologie de l'Apprentissage,
de la Mémoire & de la Communication,
NAMC, CNRS UMR 8620, Bât 446
Université Paris-Sud
91405 Orsay Cedex, FRANCE
Email benoit.delatour <@t> ibaic.u-psud.fr
Web http://www.namc.u-psud.fr