Differential Staining of Live and Dead Embryos using Fluorescein Diacetate

Procedures obtained from Ann Croy ( Guelph , Canada ) as a modification of procedures detailed in Transplantation 1971 12:148-151  Takasugi , M. An improved fluorochromatic cytotoxic test. Note that the procedure can be used for staining of all cells and not just embryos or oocytes .  Also, ethididium bromide can be replaced with propidium iodide, DAPI or Hoescht 33342

 

 

 

 

 

 

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 μ l EtBr Stock (0.05 mg/mL )

 

   3 μ l 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 μ l of dye solution on a glass slide.

 

 

 

 

  5)      In the smallest volume possible transfer embryos or oocytes to be stained in the 50 μ l 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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