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 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