APPEL at UONEURO.uoregon.edu
Fri Nov 10 15:55:24 EST 1995
I'm having trouble posting, so I thought I'd email. Post it if you think
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
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
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.
Institute of Neuroscience
1254 University of Oregon
Eugene, OR 97403
appel at uoneuro.uoregon.edu
Many thanks, Bruce!!
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