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        Differential Staining of Live and Dead Embryos using Fluorescein Diacetate

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        1026

        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

         

        <center> <p>  </p> </center>
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