פלאש מקפיא Cryosectioning E12.5 מוח עכבר

Published 5/28/2007
13 Comments
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Summary

הפגינו וידאו זו הן טכניקות פלאש מקפיא חתך ברקמת המוח העובריים של העכבר. טיפים שימושיים לשימוש cryostat מקבלים, כולל שיטות לפתרון בעיות שניתן להשתמש בהם בעת חיתוך על מנת להבטיח כי הסעיפים רקמות כתוצאה חופשיים של סדקים ועיוותים אחרים.

Cite this Article

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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. תיקון רקמות paraformaldehyde 4% PBS בפעם הרצוי.
  2. להשרות סוכרוז רקמות (cryoprotection)
    1. בצע פתרון סוכרוז 30% PBS w / v בצינור 2059.
    2. יש לשטוף את רקמת 3x ב PBS (~ 5 דקות עם נדנדה).
    3. מקום הרקמה בתמיסה סוכרוז 30%. רקמות לא תטבע.
    4. מניחים את הרקמה 4 ° C במשך הלילה, או עד שהוא שקע.
  3. גודל התווית המתאימה cryomold עם מידע והתמצאות.
  4. מלאו cryomold עם אוקטובר (למנוע בועות).
  5. העברת רקמות אמבטיה אוקטובר ומעיל עם אוקטובר
  6. העברת רקמות אוקטובר ב cryomold.
  7. המזרח הרקמה תחת מיקרוסקופ.
  8. יוצקים חנקן נוזלי לתוך צלחת פטרי מפלסטיק.
  9. במהירות ובזהירות להוריד את הרקמה cryomold בחנקן. (לא להטביע את החלק העליון של cryomold).
  10. אוקטובר כאשר הוא מוצק לבן, במקום הרקמה הקפואה אל תוך -80 ° C במקפיא לאחסון.
  11. לאזן רקמות ~ 20 מעלות צלזיוס למשך 30 דקות לפחות. לפני חתך.

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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

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

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