DETECTION OF APOPTOTIC CELLS BY ANNEXIN-V LABELING AT ELECTRON-MICROSCOPY

Annexin V is a Ca++-binding protein which is widely used as a marker f or apoptotic cells, as it binds to the phosphatidylserine residues exp osed at the surface of apoptotic cells. In this paper, we describe a m ethod for the immunogold-labeling of biotin-conjugated Annexin V, to d etect apoptotic thymocytes at electron microscopy. Etoposide-treated t hymocytes were reacted in tissue culture medium with biotin-conjugated Annexin V, fixed with glutaraldehyde, and processed for resin embeddi ng; thin sections were incubated with antibiotin antibodies coupled wi th colloidal gold. Cytometric estimates of the apoptotic index were al so performed by evaluating either the DNA content after propidium iodi de staining or the light-scattering values, as well as the positivity for fluorescein isothiocyanate-conjugated anti-biotin antibodies. At e lectron microscopy, gold-labeling of Annexin V was located on the plas ma membrane only of apoptotic thymocytes and on cytoplasmic debris, li kely resulting from the typical apoptotic blebbing. Unlabeled thymocyt es always showed normal, non-apoptotic nuclear morphology. The applica tion of Annexin V labeling at electron microscopy will allow a more re fined description of the morphological events occurring during apoptos is.