| Laboratory of P.J. Hansen,Dept. of Animal Sciences, University of Florida
http://www.animal.ufl.edu/hansen/Protocols/caspase enzyme activity.htm
 Principle
PhiPhiLux G1 D2 is a fluoroprobe that incorporates the group II caspase-recognition sequence DEVD into a bifluorophore-derivated peptide that mimics the structural loop conformation present in native protease cleavage sites. Group II caspases include caspase 3, caspase 2, and caspase 7. In this molecule, the core peptide, GDEVDGI, is coupled to a molecule of rhodamine on each side of the cleavage site. The two rhodamines interact as a dimer and emit a stable blue-green fluorescence. Cleavage of the substrate disrupts this interaction between rhodamine moieties to result in enhanced green fluorescence (excitation peak 490 nm and emission peak 520). See www.phiphilux.com for more details.
Materials
PhiPhiLux G1 D2 : OncoImmunin, Inc. (Gaithersburg, MD). Their website has lots of details
Hepes-TALP (http://www.animal.ufl.edu/hansen/ivf/mediaprep.htm#TALP)
Microscope slides: dip the slides in 1:10 poly-l-lysine solution (Sigma P8920) for 2 minutes. Allow the slides to dry. Use a marker to make a circle underneath the slide and then use a hydrophobic pen to make several circular layers on the top of the slide. These layers forms a thick circle that prevents the cover slip from damaging the embryos.
Procedure
1- Remove the embryos from culture medium and wash three times in 50-m
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