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 JoVE Biology

Flash Freezing and Cryosectioning E12.5 Mouse Brain

1, 1

1Department of Developmental and Cell Biology, University of California, Irvine (UCI)

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    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.

    Date Published: 5/28/2007, Issue 4; doi: 10.3791/198

    Cite this Article

    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

    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.

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    Disclosures

    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

    Comments

    12 Comments

    great! thanks a lot.
    Reply

    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
    Reply

    Posted by: AnonymousMay 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
    Reply

    Posted by: AnonymousJuly 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?
    Reply

    Posted by: AnonymousJuly 8, 2009, 8:18 PM

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

    Posted by: AnonymousJuly 9, 2009, 10:32 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?
    Reply

    Posted by: AnonymousJuly 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?
    Reply

    Posted by: AnonymousSeptember 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.
    Reply

    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.
    Reply

    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) 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

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