Thu Nov 9 16:44:19 EST 1995
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.
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
11. Rinse several times, then wash 4X, 15 minutes each in PBS-D-Tw, rocking or
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
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
16. Rinse several times in PBS-D. Incubate 5 minutes in DAB in PBS-D, then add
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!
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