[Histonet] fixation for frozen tissue
Serrano del Pozo, Erika
ESerrano <@t> imim.es
Tue Apr 20 02:02:40 CDT 2004
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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