Flash Congelamento e criosezionamento E12.5 cervello di topo

Published 5/28/2007
13 Comments
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Summary

Dimostrato in questo video sono le tecniche per flash congelamento e sezionamento del tessuto embrionale del cervello di topo. Consigli utili per l'utilizzo del criostato sono dati, compresi i metodi di risoluzione dei problemi che possono essere utilizzati durante il taglio per assicurare che le sezioni risultanti tessuti sono privi di crepe e altre distorsioni.

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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. Fissare il tessuto in paraformaldeide 4% in PBS per il tempo desiderato.
  2. 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.
  3. Dimensione etichetta appropriata cryomold con informazioni e orientamento.
  4. Riempire cryomold con OCT (evitare bolle).
  5. Trasferimento di tessuto a bagno ottobre e il cappotto con ottobre
  6. Trasferimento di tessuto da ottobre a cryomold.
  7. Orientare il tessuto al microscopio.
  8. Versare l'azoto liquido in plastica piastra di Petri.
  9. Rapidamente e con attenzione abbassare il tessuto cryomold in azoto. (Non immergere la parte superiore del cryomold.)
  10. Quando l'OCT è solido bianco, posto il tessuto congelato nel freezer -80 ° C per la conservazione.
  11. Equilibrare tessuto a ~ 20 ° C per almeno 30 min. prima di sezionamento.

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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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