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The popular triple fluorophore combination of MitoTracker Red CMXRos, BODIPY FL conjugated to phalloidin, and Hoechst 33258 was used to label an adherent log phase culture of HJ1.Ov cells for mitochondria, the filamentous actin network, and nuclear DNA. The cells were first treated with MitoTracker Red CMXRos in growth medium for one hour, washed and fixed with paraformaldehyde (prepared in growth medium), permeabilized, and blocked with bovine serum albumen. The cells were subsequently labeled with the conjugated phalloidin and counterstained with the bisbenzimide reagent.
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