A novel embedment-free immunoelectron microscopy technique reveals association of apoptosis-regulating proteins with subcellular structures.

By employing two electron microscopy techniques, postembedding double- and triple immunocytochemical gold-labelling combined with embedment-free electron microscopy (EF-EM), we have detected previously unreported nuclear and cytoplasmic complexes between different proapoptatic proteins in a human cancer cell line COLO 205 stimulated to apoptosis by nimesulide, a specific cyclooxygenase-2 inhibitor. Experiments with the use of double- and triple immunolabelling visualized the colocalization of proapoptotic proteins such as Bax with Bid, Bax with Bid and voltage-dependent anion channel protein (VDAC-1), and Bax with Bid and caspase-8, on organellar membranes and within the nucleus. Application of this technique in combination with EF-EM technique augments our knowledge on the precise identification and relationship of subcellular structures containing Bax, Bid, VDAC-1 and caspase-8.