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)
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Procedures
obtained from Ann Croy (
Materials
Ethidium
Bromide (EtBr; Fisher)
Fluorescein Diacetate (FDA; Sigma)
DPBS
Acetone
Procedure
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).
Live Stain=Green
Dead Stain=Red/Orange
Count green first before it
"burns out" from illumination
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