[3dem] high pressure freezing of nervous tissue

[Benoit Zuber]

Hi  Rana,

As far as brain tissue is concerned, the HPM 010 and the EMPACT / 
EMPACT2 are equivalent. Neither can vitrify brain without the use of 
cryoprotectant.

See our paper for a protocol (PNAS 2005, 102, 19192-19197). 20% dextran, 
the usual extracellular cryoprotectant used to achieve vitrification 
with high pressure freezing, is not sufficient in the case of brain. 
Some more sucrose must be added, presumably to slightly concentrate 
cytoplasm by dragging some water off the cells by osmotic pressure.

I have not tried HPM100 but in my eyes there you should not expect a 
different situation: the principles of the machine is the same as HPM010.

For EMPACT and EMPACT2, I use 200 um membrane carriers; for HPM010, I 
make a sandwich with two type-A carriers of a total thickness of 300 um.

Good luck,
Ben

-- 
Benoît Zuber
MRC Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 0QH
United Kingdom
+44 1223 402209
bzuber at mrc-lmb.cam.ac.uk



Rana Ibrahim wrote:
>
>     Hi,
>
>  
>
>     I did 2 tests of high pressure freezing (Bal-Tec HPM 010 machine) 
> followed by cryosubstitution on the olfactory bulb unfortunately the 
> biological material was full of freezing artifacts due to the high 
> water content of the nervous tissue.
>
>  
>
>     Our lab has the opportunity to use the LEICA EM HPM 100 high 
> pressure freezing machine. I am wondering whether this machine would 
> give me better freezing results on my specimens. My question is, does 
> the HPM gives better cryofixation and what are their advantages and 
> specificity relating to the other high pressure freezing machines, for 
> ex. LEICA EM-PACT 2?
>
>  
>
>     Thank you in advance,
>
>  
>
>     Rana   
>  
>
>  
>  
>  
>  
>  
> Rana Ibrahim
> Université Pierre et Marie Curie
> UMR CNRS 7102 - Neurobiologie des Processus Adaptatifs
> Bât. B - 6ème étage
> 9 quai St Bernard - 75005 Paris
> Bureau: (01) 44 27 25 65
> cell: (06) 22 80 98 50
> Adresse de messagerie: rana.ibrahim at snv.jussieu.fr 
> <mailto:rana.ibrahim at snv.jussieu.fr>
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