플래시 동결 E12.5 마우스 브레인 Cryosectioning

Published 5/28/2007
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Summary

플래시 마우스 배아 뇌 조직을 동결 sectioning위한 방법은 다음과 같습니다이 비디오에서 보여주었다. 그라 이오 스탯를 사용하기위한 유용한 팁을은 결과 조직의 섹션 균열 및 기타 왜곡 무료로되는 것을 보장하기 위해 절단하는 동안 사용할 수있는 문제 해결 방법을 포함하여 제공됩니다.

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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. 원하는 시간을 PBS에 4 % paraformaldehyde에 조직을 수정.
  2. 자당 달이다 조직 (cryoprotection)
    1. 2,059 튜브 PBS W / V에서 30 % 자당 솔루션을 확인하십시오.
    2. PBS에서 조직 배 린스 (락을 함께 ~ 5 분).
    3. 30 % 자당 용액에 넣어 조직. 조직은 침몰하지 않습니다.
    4. 그것이 침몰했다 ° C 야간, 또는 때까지 4 조직을 놓으십시오.
  3. 라벨 적절한 크기는 정보와 오리 엔테이션과 함께 cryomold.
  4. (거품을 방지) OCT와 cryomold를 입력합니다.
  5. OCT 목욕 및 OCT로 코팅을하는 조직을 전송
  6. cryomold에서 OCT하기 위해 조직을 전송합니다.
  7. 현미경 동양의 조직.
  8. 플라스틱 페트리 접시에 액체 질소를 부어.
  9. 신속하고 조심스럽게 질소로 cryomold의 조직을 낮추십시오. (잠수함 cryomold의 상단을하지 않습니다.)
  10. OCT는 흰색 고체 경우 -80 ° C 저장 냉동실에 냉동 조직을 놓으십시오.
  11. ~ 20 ° C 이상에서 30 분 조직을 평형. 이전 sectioning 수 있습니다.

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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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