Differential Staining of Live and Dead Embryos using Fluorescein Diacetate

and Ethidium Bromide

J. Lannett Edwards and P.J. Hansen

Dept. of Animal Sciences, University of Florida

(JLE is now at University of Tennessee)

List of Other Protocols  |  P.J. Hansen Home Page   |  Dept .of Animal Sciences

University of Florida

 

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

 

 

Page maintained by P.J. Hansen        hansen@animal.ufl.edu        http://www.animal.ufl.edu/hansen        Protocol created: February 2, 1995; online 02-21-2005        Last updated: February 22, 2005        Original material © J. Lannett Edwards and Peter J. Hansen        Links to commercial sites do not constitute endorsement by the         authors or the University of Florida.

University of Florida Department of Animal Sciences PO Box 110910 Gainesville, Florida 32611 Phone:(352) 392-5590 Fax:(352) 392-3922