BrdU labeling

[Bruce Appel]

Hi Lisa,

I'm having trouble posting, so I thought I'd email. Post it if you think 
it's useful.

Here's a protocol we've used on <24 h embryos. I believe others around 
here have used it on considerably older (larval stage) animals. There's 
a bit in here about RNase-free conditions and long fixation times-that's 
because we were using it with in situ RNA hybridization. If you are just 
doing the BrdU bit, I imagine a 2 hr fix, as if for antibody staining, 
would be adequate. We did the RNA hybridization on whole embryos, 
sectioned them, and stained for BrdU on slides. Anyway, the upshot is 
that there is no need for injection, just let them soak in it. Good 
luck.
Bruce


Embryos of each stage were hand-dechorionated and placed for thirty 
minutes in a 6(infinity) C ice water bath in a solution of 10 mM BrdU/15% DMSO in 
Ringer's. At thirty minutes, embryos were quickly rinsed three times 
with Ringer's.  Embryos from each stage were then divided equally in to 
two groups;  either a 15 minute group or a 30 minute group.  Embryos 
were then placed in Ringer's in a covered 35 ml petri dish on a warm 
plate at 28.5(infinity) C to resume their development for the designated 15 or 30 
additional minutes.
   At the end of both time periods, embryos were anesthetized with 
tricaine and then placed in RNase-free 4% PFA in scintillation vials.  
The embryos in fix were placed on a rotator at room temperature for 12 
to 24 hours, and rinsed three times with RNase-free 1XPBS (first rinse 
fast, last two rinses five minutes on a rotator).  Embryos were then 
rinsed three times five minutes each in 100% MeOH and stored at 0(infinity) C.

Staining:  Slides were incubated in 2N HCl for 60 minutes at 37(infinity) C (to 
expose the DNA) and then rinsed five times over ten minutes in 1X PBS.  
Slides were then incubated in anti-BrdU (an mIgG1; Boehringer Mannheim) 
at 1/50 in PBDT 2% normal goat serum for 2 hours at room temperature.  
They were then rinsed three times over ten minutes in 1X PBS/TX, 
incubated for one hour at room temperature  in gam IgG TRITC at 1/200 in 
PBDT with 5% ngs, and went through a final rinse of three times over ten 
minutes in 1X PBS.  Slides were coverslipped using two to three drops of 
PBS/glycerol (1:1) per slide.
Bruce Appel
Institute of Neuroscience
1254 University of Oregon
Eugene, OR 97403
appel at uoneuro.uoregon.edu

Many thanks, Bruce!!

Lisa