Flash de congelación y muestras criostáticas E12.5 cerebro de ratón

Published 5/28/2007
13 Comments
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Summary

Se demuestra en este vídeo son las técnicas de congelación para el flash y el corte del tejido cerebral de embriones de ratón. Consejos útiles para el uso del criostato se les da, incluyendo los métodos de solución de problemas que se pueden utilizar durante el corte para asegurar que las secciones de tejidos resultantes están libres de grietas y otras distorsiones.

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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. Fijar el tejido en el 4% paraformaldehido en PBS durante el tiempo deseado.
  2. Sacarosa infundir tejido (crioprotector)
    1. Hacer 30% de solución de sacarosa en PBS w / v en 2059 tubo.
    2. Enjuague el tejido 3 veces en PBS (~ 5 min con la oscilación).
    3. Coloque el tejido en una solución de sacarosa al 30%. Tejido no se hunda.
    4. Coloque el tejido en 4 ° C durante la noche, o hasta que se ha hundido.
  3. Tamaño de la etiqueta apropiada cryomold con información y orientación.
  4. Rellene con cryomold octubre (evitar las burbujas).
  5. La transferencia de tejido para baño octubre y cubrir con octubre
  6. La transferencia de tejido para octubre en cryomold.
  7. Orientar el tejido al microscopio.
  8. Vierta el nitrógeno líquido en placa Petri de plástico.
  9. De forma rápida y cuidadosamente baje el tejido en cryomold en el nitrógeno. (No sumergir la parte superior de la cryomold.)
  10. Cuando el OCT es un sólido blanco, coloque el tejido congelado en -80 ° C congelador para el almacenamiento.
  11. Equilibrar los tejidos a ~ 20 ° C durante al menos 30 min. antes de cortar.

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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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