Flash Congelamento e criosezionamento E12.5 cervello di topo (Translated to Italian)

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Cite this Article: Flash Congelamento e criosezionamento E12.5 cervello di topo

Currle, D. S., Monuki, E. S. Flash Freezing and Cryosectioning E12.5 Mouse Brain. J. Vis. Exp. (4), e198, doi:10.3791/198 (2007).

Protocol: Flash Congelamento e criosezionamento E12.5 cervello di topo

Fissare il tessuto in paraformaldeide 4% in PBS per il tempo desiderato.
Saccarosio infondere tessuto (crioprotezione)
1. Preparare la soluzione di saccarosio al 30% in PBS w / v nel 2059 tubo.
2. Lavare il tessuto in PBS 3x (~ 5 min con dondolo).
3. Tessuto posto in soluzione di saccarosio al 30%. Tessuto non affonderà.
4. Posizionare il tessuto in 4 ° C durante la notte, o fino a quando è affondata.
Dimensione etichetta appropriata cryomold con informazioni e orientamento.
Riempire cryomold con OCT (evitare bolle).
Trasferimento di tessuto a bagno ottobre e il cappotto con ottobre
Trasferimento di tessuto da ottobre a cryomold.
Orientare il tessuto al microscopio.
Versare l'azoto liquido in plastica piastra di Petri.
Rapidamente e con attenzione abbassare il tessuto cryomold in azoto. (Non immergere la parte superiore del cryomold.)
Quando l'OCT è solido bianco, posto il tessuto congelato nel freezer -80 ° C per la conservazione.
Equilibrare tessuto a ~ 20 ° C per almeno 30 min. prima di sezionamento.

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Disclosures: Flash Congelamento e criosezionamento E12.5 cervello di topo

Materials: Flash Congelamento e criosezionamento E12.5 cervello di topo

Name	Company	Catalog Number	Comments
Tissue-Tek Cryomold	Ted Pella, Inc.	27181
O.C.T.	Ted Pella, Inc.	27050
Sucrose solution			30% sucrose solution in PBS w/v
paraformaldehyde			4% paraformaldehyde in PBS

Ask the Author: Flash Congelamento e criosezionamento E12.5 cervello di topo

11 Comments

This is one application almost all labs use either for some immuno or insitu experiments and seeing the procedure to this level of detail, I think will enable people to achieve better results with less trial and error.

Posted by: AnonymousJuly 6, 2007, 9:41 PM

awesome

Posted by: AnonymousJuly 18, 2007, 10:42 PM

great! thanks a lot.

Posted by: AnonymousSeptember 25, 2007, 2:01 PM

The presentation was wonderful and I learned more from this video than what my conservative collegues in the lab explained me.

Thanks a lot

Posted by: #3May 29, 2008, 4:58 PM

I'm having some issues washing off the OCT with BPS from the slides. - I did some retrograde staining of peripheral nerves and I cross sectioned the spinal cord of mice-
the problem is that as I add PBS drop by drop, or as I place the slide into plate with PBS liquid layer my samples keep falling off the slide..
Any ideas? because I'm running out of them
e-mail me please: jccs_85@hotmail.com

Posted by: CarlosJuly 2, 2009, 9:35 AM

This is a great video demonstrating how to cryosection brain that will certainly help me with my future studies. I was wondering, is it always necessary and/or desirable to fix the brain with paraformaldahyde prior to cryoprotecting with sucrose? I seem to recall hearing for some applications, such as IHC, paraformaldahyde fixation may disrupt the antibody/antigen interaction for certain antibodies used. Would someone be willing to comment on this?

Posted by: Shelly July 8, 2009, 8:18 PM

send me an e-mail at spencer.currle@stjude.com

6.1

Posted by: AnonymousJuly 9, 2009, 10:32 AM

Posted by: Shelly July 9, 2009, 4:42 PM

I have the same question as Shelly. Some applications require fixing tissue after cryosectioning. Do you have a protocol for this?

Posted by: SueSeptember 20, 2010, 3:03 PM

Hello, my name is Xiang Weng, I am a student in Long Island University, department of Biomedical Science, I want to learn cryosectioning, thank you.

Posted by: Xiang W.October 5, 2012, 11:24 AM

Hello, my name is Xiang Weng, I am a student in Long Island University, department of Biomedical Science, I want to learn cryosectioning, thank you.

Posted by: Xiang W.October 5, 2012, 11:35 AM

I have looked all over for the answer but I cannot find it: How many seconds (or how long in general) does it take to flash freeze a serum lab sample using ethanol and dried ice? Thanks!

Posted by: Michele K.April 3, 2013, 8:51 PM

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Date Published	5/28/2007
JoVE Section	General
JoVE Issue	4
Comments	11 Comments
View Count	Loading... (Details…)
DOI	doi:10.3791/198

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Flash Congelamento e criosezionamento E12.5 cervello di topo