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Flash Freezing and Cryosectioning E12.5 Mouse Brain

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

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Cite this Article: Flash Freezing and Cryosectioning E12.5 Mouse Brain

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: Flash Freezing and Cryosectioning E12.5 Mouse Brain

  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: Flash Freezing and Cryosectioning E12.5 Mouse Brain

Materials: Flash Freezing and Cryosectioning E12.5 Mouse Brain

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

Ask the Author: Flash Freezing and Cryosectioning E12.5 Mouse Brain

11 Comments

This is one application almost all labs use either for some immuno or insitu experiments and seeing the procedure to this level of detail, I think will enable people to achieve better results with less trial and error.

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Posted by: AnonymousJuly 6, 2007, 9:41 PM

awesome

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Posted by: AnonymousJuly 18, 2007, 10:42 PM

great! thanks a lot.

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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

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Posted by: #3May 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

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Posted by: CarlosJuly 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?

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Posted by: Shelly July 8, 2009, 8:18 PM

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

6.1

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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?

7

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Posted by: Shelly July 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?

8

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Posted by: SueSeptember 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.

9

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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.

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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) does it take to flash freeze a serum lab sample using ethanol and dried ice? Thanks!

11

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Posted by: Michele K.April 3, 2013, 8:51 PM

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