RE: [Histonet] fresh-frozen mouse tail sectioning

From:"Rachel, Rivka (NIH/NEI) [E]"

Do you have to freeze or section the tissue at all?  If there is significant beta-galactosidase activity, you should be able to just put the tail tip in X-gal and see if it turns blue, having of course appropriate positive and negative control tails that are known to carry or not carry, respectively, the relevant transgene.


Rivka A. Rachel, MD, PhD
Staff Scientist, National Eye Institute
Neurobiology-Neurodegeneration and Repair Laboratory
Tel: 301 443-4906

-----Original Message-----
From: Gaupp, Dina D  []
Sent: Fri 5/16/2008 12:04 PM
Subject: [Histonet] fresh-frozen mouse tail sectioning
To Whomever This Applies:
I am having big problems sectioning mouse tail, approximately 2mm in thickness, vertical embedded in OCT for frozen sections.  The tissue is fresh-frozen because the principle investigator would like to detect an enzyme x-gal.  No fix, no cryopreservation - the tissue was not immersed in any type of solution.  He snipped the ends of a mouse tail & immediately gave me the tissues.  I embedded the tissue vertically in OCT & flash froze at -80C.  Upon sectioning, the tissue rolled.  I could not for the life of me get 1 section.  All the tissues rolled & its so tiny to begin with that it was hard for me to grab it.  I used the anti-roll plate, hoping it would hold the tissue in place but it still rolled.  I changed temperature settings(increase & decrease temp), thickness, angle, rubbed it with my fingers.  Everything I could think of to get a section.  I asked someone else in the lab to try & they couldn't get a section.  It was like the tissue completely separated from OCT.  The principle investigator will give me more samples & I don't want this to happen again.  
Can anyone in histoland help me, tell me what I didn't do or what I did wrong?
Dina D. Gaupp, BS, MT
Senior Lab Supervisor
Center for Gene Therapy, SL-99
Tulane University Health Science Center
1430 Tulane Ave
New Orleans, La 70112
Lab: 504-988-1194  
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