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.

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 
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 
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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