Many thanks to all who responded to my request for BrdU
protocols in zebrafish embryos and larva. I received several
protocols that have worked in otherlabs, and I though the
information might be helpful to others, so here are the
protocols I received:
From Steve Devoto at U. Oregon:
To: llc4q@avery.med.Virginia.EDU
Date: Thu, 9 Nov 1995 14:44:19 PDT
Subject: Re: BrdU
>To: zbrafish@net.bio.net
>From: llc4q@avery.med.Virginia.EDU (Lisa Lynn Cunningham)
>Subject: BrdU
>Date: Thu, 9 Nov 1995 20:16:27 GMT
Lisa:
I don't have time right now to elaborate on this, but this is a protocol I printed
out. I will be happy to give more comments or info later.
WHOLE MOUNT ASSAY FOR CELL PROLIFERATION
A. PBS-D-Tw: PBS + 1% DMSO + 0.1% Tween-20
B. 4% PF: 4% paraformaldehyde, 50 mM NaPO4, pH 7.4.
C. 10 mM BrdU: made up in 0.2 M KCl, with 0.2% phenol red.
C (Alternative). 10mM BrdU: made up in embryo medium with 15% DMSO
D. 2 N HCl, in water.
E. 10 mg/ml proteinase K stock.
F. Anti-BrdU antibody (e.g. Boehringer, 1170 376; diluted 1:100 in Blocking
Solution), and appropriate secondary antibody and detection system.
G. Blocking Solution: PBS-D-Tw + 1% BSA + 2% Normal Goat Serum.
Method
1. Injection (I have used this on 1 hour to 16 hour old embryos with success):
10 mM BrdU into yolk of embryo of desired age.
1 (Alternative). Soaking (I have used this on 6 hour to 72 hour old embryos with
success). Dechorionate embryo of desired age, and place in 10mM BrdU, made up in
Embryo Medium with 15% DMSO at 6 to 8 degrees for 20 minutes (I do this with petri
dish resting on top of ice).
2. Allow embryos to develop to desired age.
3. Fix in 4% PF, several hours at RT, or ON in the cold.
4. Remove PF, wash embryos 2X in methanol. Put embryos in fresh methanol, place
at -20oC, for at least an hour. Indefinite storage at -20oC is fine.
5. Rehydrate using a graded Methanol/PBS series: 75%, 50%, 25% (They sometimes
stick to sides of tubes in 25% step, I just continue). Place in PBS-Tw (if stuck,
they now come free).
6. (Optional, helps antibody penetration, doesn't seem to hurt morphology)
Digest with 10 ug/ml Proteinase K in PBS-Tw, 20 minutes at RT. Rinse several
times in PBS-Tw. Refix embryos in 4%PF for 10-30 minutes.
7. Rinse several times in water. (Note: embryos sometimes stick to sides of
tubes during this, I just continue, this would probably be prevented by including
DMSO or 0.1% Tween in the water). Wash 1 or 2 times in 2N HCl, incubate at RT in
2N HCl for 1 hour.
8. Rinse several times in PBS-Tw.
9. Incubate 10 minutes in blocking solution, rocking or shaking gently.
10. Incubate 2 hours, or more (RT), in anti-BrdU (ON in cold is fine), rocking or
shaking gently.
11. Rinse several times, then wash 4X, 15 minutes each in PBS-D-Tw, rocking or
shaking gently.
12. Incubate 2 hours or more with goat anti-mouse antibody, rocking or shaking.
13. Rinse several times, then wash 4X, 15 minutes each in PBS-D-Tw, rocking or
shaking.
14. Incubate 2 hours or more with mPAP, rocking or shaking.
15. Rinse several times, then wash 4X, 15 minutes each in PBS-D-Tw, rocking or
shaking.
16. Rinse several times in PBS-D. Incubate 5 minutes in DAB in PBS-D, then add
H2O2.
NOTES: It is also possible to do the staining on sections of embryos. I would
fix the embryos as above in 4%PF, and prepare cryostat sections. These can then
be treated with HCl, and immunostained using either HRP or fluorescent probe
conjugated secondary antibodies.
Many thanks, Steve! I'll try this one this week!
Lisa
From Daniel Alexandre:
To: llc4q@avery.med.Virginia.EDU (Lisa Lynn Cunningham)
From: d.alexandre@kcl.ac.uk (Daniel ALEXANDRE)
Subject: Re: BrdU
Date: Fri, 10 Nov 1995 11:49:31 +0000
Hello Lisa,
Below is a protocol I got from Lucy Smithers (ICRF, Oxford) who got it from
Judith Eisen (Eugene)
Judith's protocol for embryos between tailbud and 14 somites:
BrdU incorporation: Embryos of each stage were hand-dechorionated*
and placed for thirty minutes in a 6 C ice water
bath in a solution of 10 mM BrdU/15% DMSO in Ringer's (in eppendorfs). At
thirty minutes, embryos were quickly rinsed three times with Ringer's.
Embryos from each stage were then divided equally into 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 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 C.
*Dechorionated embryos were manipulated on solidified 2% agar in Ringer's in
either 30 ml dishes or in 1.5 ml eppendorf tubes (for BrdU exposure) to
avoid rupture of the embryos upon contact with a plastic surface.
Sectioning: Embryos were taken out of fix and rinsed three times in 1X PBS,
mounted in 1.5% agar/5% sucrose blocks and sunk in 30% sucrose overnight.
Blocks were frozen sectioned on a cryostat at -20 C and collected on
gel-subbed slides. Embryos of three somites and fewer were sectioned
in cross-section and embryos between six and fourteen somites were
sectioned sagitally.
Staining: Slides were incubated in 2N HCl for 60 minutes at 37 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) at 1/50 in PBDT 2% normal
goat serum (ngs) 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 goat anti-mouse IgG TRITC (or FITC) 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 (or use Citifluor).
Comment from Lucy Smithers:
Follow this closely, and it should work nicely.
PBDT is as in the zebrafish book: PBS, 1% BSA, 1% DMSO, 0.1% Triton
X-100. (TX is Triton X-100).
You don't have to section the embryos - removing the yolk from 1 - 10
somite stage embryos and flat-mounting them works just as well. Also, if
you're used to looking at transverse sections, then do those rather than
sagittal - whatever you find most helpful.
I haven't tried this method yet. If you get a protocol to label older
embryos, I'd be pleased to know about it.
Regards,
Daniel ALEXANDRE
Developmental Biology Research Centre
Randall Institute
King's College Tel: (44) (0) 171 836 8851
26-29 Drury Lane Fax: (44) (0) 171 497 9078
London WC2B 5RL e-mail: d.alexandre@kcl.ac.uk
Again, many thanks, Daniel!
Lisa
From Bruce Appel:
From: "Bruce Appel"
To: llc4q@avery.med.Virginia.EDU
Date: Fri, 10 Nov 1995 13:55:24 PDT
Subject: BrdU labeling
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@uoneuro.uoregon.edu
Many thanks, Bruce!!
Lisa