Differential Staining of Live and Dead Embryos using Fluorescein Diacetate
and Ethidium Bromide
J. Lannett Edwards and P.J. Hansen
Dept. of Animal Sciences,
(JLE is now at University of Tennessee)
Procedures obtained from Ann Croy (
Ethidium Bromide (EtBr; Fisher)
Fluorescein Diacetate (FDA; Sigma)
1) Make following stock solutions:
EtBr (10 mg/mL DPBS) Store in dark at 4 C
FDA (5 mg/mL acetone) Store in dark in glass container at -20 C
Storage life of stocks ~4 months
2) Just before use (i.e., ~10 min) prepare the following in a 15 mL conical tube covered with aluminum foil:
100 μl EtBr Stock (0.05 mg/mL)
3 μl FDA Stock (0.005 mg/mL)
10 mL DPBS or culture medium
Note: If you want to recover embryos from glass slide, use DPBS with 0.1% BSA or serum to prevent them from sticking to slide.
3) Place on ice until needed.
4) To stain embryos or oocytes place 50 μl of dye solution on a glass slide.
5) In the smallest volume possible transfer embryos or oocytes to be stained in the 50 μl and allow to sit in the dark for at least 3 min (FDA cleavage of acetate radical traps dye inside cell; 3 min=time for accumulation).
6) View embryos or oocytes for staining using the fluorescence microscope under UV epiluminesence (use UV filter).
Count green first before it "burns out" from illumination
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Original material © J. Lannett Edwards and Peter J. Hansen
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