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פלאש מקפיא Cryosectioning E12.5 מוח עכבר

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

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Cite this Article: פלאש מקפיא Cryosectioning E12.5 מוח עכבר

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: פלאש מקפיא Cryosectioning E12.5 מוח עכבר

  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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Disclosures: פלאש מקפיא Cryosectioning E12.5 מוח עכבר

Materials: פלאש מקפיא Cryosectioning E12.5 מוח עכבר

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: פלאש מקפיא Cryosectioning E12.5 מוח עכבר

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.

1

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

awesome

2

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

great! thanks a lot.

3

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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

4

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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

5

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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?

6

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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.

10

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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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