RE: [Histonet] In vivo fluorescent antibody immunocytochemistry

From:"Liz Chlipala"


We have not looked at antibodies before, but we have looked at FITC and Cy3
tagged compounds.  When we did this we looked at different time points up to
24 hours.  You could use the liver and kidney as possible controls, once
injected the antibody will be metabolized through the kidney and liver.  We
would snap freeze the tissue and then cut frozen sections.  In our
application we had some issues with fixation since our compound was water
soluable.  But we found that brief fixation in 10% NBF preserved the
staining, but we also found that increased time in buffers or water, etc.
would decrease staining.  Personally I would start with a pilot of a few
animals. The time points that you select will also depend it they are dosing
iv verses ip or sc.  Its going to take longer for ip and sc dosing to
metabolize than the iv.  I would try 1, 4, 8 and 24 hours to start with.
After you cut the frozen don't mount them, just look at the section under
the fluorescent microscope, you should see fluorescence in the kidney at all
of those time points.  You will get an idea of what type of staining you
have and then you can fix and mount and be able to judge if you are losing
staining when you fix, etc.

Good Luck


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
P.O. Box 18592
Boulder, CO 80308
phone (303) 735-5001
fax (303) 735-3540
Ship to Address:
Premier Laboratory, LLC
University of Colorado at Boulder
MCDB, Room A3B40
Boulder, CO 80309

-----Original Message-----
[] On Behalf Of Johnson,
Sent: Friday, May 04, 2007 10:30 AM
Subject: [Histonet] In vivo fluorescent antibody immunocytochemistry

Has anybody successfully done experiments by injecting
fluorescent-labeled antibodies into mouse organs in vivo? I'm currently
searching the internet looking for information on how this is done, how
long these antibodies would need to "incubate" prior to sacrifice, and
whether it is possible to detect them in histological section

Thank you for any insight you can provide!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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