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

• Published in: Journal of molecular histology 2002-08, Vol.34 (8-9), p.441-446
• Main Authors: Gajkowska, B, Wojewódzka, U
• Format: Article
• Language: English
• Subjects: Adenocarcinoma - metabolism; Adenocarcinoma - ultrastructure; Anion channels; Apoptosis; Apoptosis - drug effects; Apoptosis - physiology; Bax protein; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; BID protein; Cancer; Carrier Proteins - metabolism; Caspase 8; Caspase 9; Caspases - metabolism; Cell Membrane - ultrastructure; Cell Nucleus - metabolism; Cell Nucleus - ultrastructure; Cells; Cellular Structures - metabolism; Cellular Structures - ultrastructure; Colonic Neoplasms - metabolism; Colonic Neoplasms - ultrastructure; Cyclooxygenase 2; Electron microscopy; Humans; Immunoelectron microscopy; Isoenzymes - antagonists & inhibitors; Membrane Proteins; Microscopy; Microscopy, Immunoelectron - methods; Mitochondria - metabolism; Mitochondria - ultrastructure; nimesulide; Nuclear Envelope - ultrastructure; Nuclei; Porins - metabolism; Porins - ultrastructure; Prostaglandin-Endoperoxide Synthases; Proteins; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-bcl-2; Staining and Labeling; Sulfonamides - pharmacology; Tumor cell lines; Tumor Cells, Cultured; Voltage-Dependent Anion Channels
• ISSN: 0018-2214; 1567-2379; 1567-2387; 1573-6865
• DOI: 10.1023/A:1023643722886

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.