Flash Freezing and Cryosectioning E12.5 Mouse Brain

Published 5/28/2007
13 Comments
  CITE THIS  SHARE 
Biology

You must be subscribed to JoVE to access this content.

Fill out the form below to receive a free trial:

Welcome!

Enter your email below to get your free 10 minute trial to JoVE!





By clicking "Submit," you agree to our policies.

 

Summary

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Cite this Article

Copy Citation

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

Abstract

Protocol

  1. Fix tissue in 4% paraformaldehyde in PBS for desired time.
  2. Sucrose infuse tissue (cryoprotection)
    1. Make 30% sucrose solution in PBS w/v in 2059 tube.
    2. Rinse tissue 3x in PBS (~ 5 min with rocking).
    3. Place tissue in 30% sucrose solution.  Tissue will not sink.
    4. Place the tissue in 4°C overnight, or until it has sunk.
  3. Label appropriate size cryomold with information and orientation.
  4. Fill cryomold with O.C.T. (avoid bubbles).
  5. Transfer tissue to O.C.T. bath and coat it with O.C.T.
  6. Transfer tissue to O.C.T. in cryomold.
  7. Orient the tissue under microscope.
  8. Pour liquid nitrogen into plastic Petri dish.
  9. Quickly and carefully lower the tissue in cryomold into the nitrogen. (do not submerge the top of the cryomold.)
  10. When the O.C.T. is solid white, place the frozen tissue into -80°C freezer for storage.
  11. Equilibrate tissue to ~20°C for at least 30 min. prior to sectioning.

Subscription Required. Please recommend JoVE to your librarian.

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

DOWNLOAD MATERIALS LIST

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

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Video Stats