Katryn Sunn wrote:
To: fluorpro at net.bio.net
From: k.sunn at garvan.unsw.edu.au (Kathryn Sunn)
Subject: COLOCALISATION WITH GFP
Date: 10 Oct 1999 22:52:34 -0700
Dear All
Has anyone done a lot of colocalisation studies using GFP tagged
transfected constructs and a red-tagged antibody? If so has anyone ever had
the problem of the GFP green signal losing its fluorescence after
permeabilisation? Does anyone know any handy hints to get around this at
all?
Thanks for your help
Kate
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Kathryn Sunn BSc (Hons)
Postgraduate Student
Bone and Mineral Research Program
Garvan Institute of Medical Research
384 Victoria St, Darlinghurst
St Vincent's Hospital Tel: (02) 9295 8260
Sydney NSW 2010 Fax: (02) 9295 8241
Australia email: k.sunn at garvan.unsw.edu.au
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Hi Katryn
It proabely depends on your procedure. Whitch procedure are you using?
I have immunostained the transferrin receptor with texas-red in GFP-fusion
expressing primary muscle cells, with out any problems with the EGFP
fluorescence. I use fist 2% paraformaldehyde to fix followed with saponin
for permeabilisation and finally the antibodies with saponin. I mount in
vectashield. I have not seen any decrease in EGFP fluorescence on specimen
even after 1½ year in the fridge.
Med venlig hilsen Hans
Hans P.M.M.Lauritzen, B.Sc., M.Sc., Phd-student.
Copenhagen Muscle Research Center, The Panum Institute 12.4
Copenhagen, Denmark
E-mail: H.P.Lauritz at mfi.ku.dk
Phone: +45 35327375 or +45 35327436 or +45 35327430
Fax: +45 35327420
