Annexin V is a Ca(++)-binding protein which is widely used as a marker for 
apoptotic cells, as it binds to the phosphatidylserine residues exposed at the 
surface of apoptotic cells. In this paper, we describe a method for the 
immunogold-labeling of biotin-conjugated Annexin V, to detect apoptotic 
thymocytes at electron microscopy. Etoposide-treated thymocytes were reacted in 
tissue culture medium with biotin-conjugated Annexin V, fixed with 
glutaraldehyde, and processed for resin embedding; thin sections were incubated 
with antibiotin antibodies coupled with colloidal gold. Cytometric estimates of 
the apoptotic index were also performed by evaluating either the DNA content 
after propidium iodide staining or the light-scattering values, as well as the 
positivity for fluorescein isothiocyanate-conjugated anti-biotin antibodies. At 
electron microscopy, gold-labeling of Annexin V was located on the plasma 
membrane only of apoptotic thymocytes and on cytoplasmic debris, likely 
resulting from the typical apoptotic blebbing. Unlabeled thymocytes always 
showed normal, non-apoptotic nuclear morphology. The application of Annexin V 
labeling at electron microscopy will allow a more refined description of the 
morphological events occurring during apoptosis.