[Histonet] fixation for frozen tissue

[Serrano del Pozo, Erika]

Hi Rita,

I only use fixation before freeze a tissue when I can´t handle it when it´s
fresh (diaphragm), and I perform a sucrose treatment after the fixation.

Here is what I do:

fixation:
paraformaldehid 4% at 4ºC (minimum time o/n) 

fast washed in PBS 0,1M 

criproteccion (I):  
sucrose 15% (75g sucrose - 1ml NaN3 10% - to 500ml PBS) o/n at 4ªC; shaking 

crioproteccion (II):  
sucrose 30% (150g sucrose - 2ml NaN3 10% - to 1000ml PBS) shaking at 4ªC;
several changes (minimum 3) until the samples are at heart deposited of the
container (I don´t know why, but this not always happens; don´t worry, it
also works)
 
fast washed in PBS 0,1M 

frezee in OCT with liquid nitrogen

Wishing you a "sweet" procedure. 


Erika Serrano

Centre de Regulació Genòmica
Differenciation and Cancer Programme
Barcelona -SPAIN-

> ----------
> From: 	Rita Angel
> Sent: 	Monday, April 19, 2004 19:24 PM
> To: 	histonet <@t> lists.utsouthwestern.edu
> Subject: 	[Histonet] fixation for frozen tissue
> 
> Hi all,
> 
> I have several questions about fixing frozen tissue. In the past, we've 
> always frozen tissue fresh from the animal into frozen embedding media and
> 
> then immersing into liquid nitrogen.
> 
> I have an investigator that brought frozen tissue that he first fixed in 
> formalin, then froze. I'm unable to get sections because the tissue is 
> soft. It seems there is still moisture in the tissue, so I melted the
> block 
> & blotted off the tissue, then re-froze. I'm still not able to get
> sections 
> although it doesn't seem as mushy now. It still seems like the tissue is 
> wet.Does anyone have any suggestions?
> 
> He was also asking about a protocol for sucrose, and I was wondering if 
> anyone could get one to me? Also why do people use this procedure, and
> when 
> do you need to use it?
> 
> Thanks for all your help,
> 
> Rita Angel, HT
> 
> 
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