[Histonet] BrdU Indirect IF staining

[Johnson, Teri]

We are attempting to do some indirect immunofluorescent double staining
with anti-BrdU antibody in mouse tissues and various other antibodies.
One of the problems we are having is denaturing the DNA for our BrdU
staining.  We obtain a strong, but somewhat non-specific signal with
DNAse digestion. HCl pretreatment renders the nuclei unstainable by
DAPI.  We have had no luck in using HIER for denaturing (electric
pressure cooker method). I suspect with most of our double stains with
BrdU, we will be required to do the other antibody first, then denature
and incubate with anti-BrdU, then use the fluorescent-labeled
secondaries for detection.
 
 
Can someone give me some suggestions as to how to optimize our
pretreatment regimen without loss of nuclear integrity for
immunofluorescence?  What are you doing for BrdU immunostaining out
there that's working, esp. using fluorescent-labeled secondaries?
 
Has anybody successfully used the Xenon antibody labeling kit for direct
labeling primary antibodies, and obtained good results in tissue IHC?
 
Thanks for your help!
 

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj <@t> stowers-institute.org