Flash de congélation et Cryosectioning E12.5 cerveau de souris

Published 5/28/2007
13 Comments
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Summary

Démonstration dans cette vidéo sont les techniques pour flash de congélation et de sectionnement des tissus cérébraux embryonnaires à partir de la souris. Conseils utiles pour l'utilisation du cryostat sont donnés, y compris les méthodes de dépannage qui peut être utilisé pendant la coupe pour s'assurer que les sections résultante tissus sont exempts de fissures et d'autres distorsions.

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

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Abstract

Protocol

  1. Fixer le tissu dans du paraformaldéhyde 4% dans du PBS pendant le temps désiré.
  2. Saccharose infuser tissus (cryoprotection)
    1. Faire solution de saccharose à 30% dans le PBS p / v en 2059 tube.
    2. Rincez le tissu 3x dans du PBS (~ 5 min avec bascule).
    3. Tissulaire lieu en solution de saccharose à 30%. Tissus ne va pas couler.
    4. Placez le tissu dans 4 ° C pendant la nuit, ou jusqu'à ce qu'il ait coulé.
  3. Taille de l'étiquette appropriée cryomold d'information et d'orientation.
  4. Remplissez cryomold avec OCT (éviter les bulles).
  5. Transfert des tissus à bain d'PTOM et l'enrober avec octobre
  6. Transfert des tissus aux PTOM cryomold.
  7. Orienter les tissus sous microscope.
  8. Verser l'azote liquide dans une boîte de Petri en plastique.
  9. Rapidement et soigneusement bas le tissu dans cryomold dans l'azote. (Ne pas immerger le haut de la cryomold.)
  10. Lorsque l'OCT est solide blanc, placez le tissu congelé en congélateur à -80 ° C pour le stockage.
  11. Equilibrer les tissus à ~ 20 ° C pendant au moins 30 min. avant la coupe.

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Materials

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

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Comments

13 Comments

  1. great! thanks a lot.

    Reply
    Posted by: Anonymous
    September 25, 2007 - 2:01 PM
  2. The presentation was wonderful and I learned more from this video than what my conservative collegues in the lab explained me. Thanks a lot

    Reply
    Posted by: Anonymous
    May 29, 2008 - 4:58 PM
  3. 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

    Reply
    Posted by: Anonymous
    July 2, 2009 - 9:35 AM
  4. 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?

    Reply
    Posted by: Anonymous
    July 8, 2009 - 8:18 PM
  5. send me an e-mail at spencer.currle@stjude.com

    Reply
    Posted by: Anonymous
    July 9, 2009 - 10:32 AM
  6. 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?

    Reply
    Posted by: Anonymous
    July 9, 2009 - 4:42 PM
  7. I have the same question as Shelly. Some applications require fixing tissue after cryosectioning. Do you have a protocol for this?

    Reply
    Posted by: Anonymous
    September 20, 2010 - 3:03 PM
  8. 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.

    Reply
    Posted by: Xiang W.
    October 5, 2012 - 11:24 AM
  9. 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.

    Reply
    Posted by: Xiang W.
    October 5, 2012 - 11:35 AM
  10. I have looked all over for the answer but I cannot find it: How many seconds (or how long in general) dŒs it take to flash freeze a serum lab sample using ethanol and dried ice? Thanks!

    Reply
    Posted by: Michele K.
    April 3, 2013 - 8:51 PM
  11. Hello, i am Kudirat, i am a student from University of Barcelona, department of Analytical Chemistry but basically I am in research area of Chemometric. Currently, we are working on a project where we want to get cryosections of Daphnia magna.
    Here are what we have done:
    1 ) We prepare the sample embedding it with 100% OCT, and flash freezing with Liquid Nitrogen directly.
    2) Finally we cut with the microtome cryostat between 12-20 um, which was not sucessful.

    What we have not done.
    1) Fix the sample. This is because we thought it might affect in our results since we are planning to acquire images with these cryosections in FT-IR Spectroscopy.

    At this moment we are planning to try chemical fixation, any suggestions would be greatly appreciated since we have little idea of biology.

    Thank you.


    Reply
    Posted by: Anonymous
    March 14, 2017 - 6:01 AM

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