Reflection confocal imaging of type I and type III isozymes of hexokinase in PC12 cells

Reflection confocal microscopy was used to determine the intracellular distribution of Type I and III isozymes of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in PC12 cells; detection was by staining with diaminobenzidine as a substrate for horseradish peroxidase-conjugated antimouse immunoglobulins bound to isozyme-specific monoclonal antibodies. With both isozymes, detection of the staining pattern was significantly enhanced by reflection confocal imaging compared with viewing with transmitted brightfield optics. For Type I, prominent staining of cytoplasmic organelles having a distribution consistent with that of mitochondria was noted. For Type III, intense staining at the nuclear periphery was observed. A distinct punctate pattern along the nuclear surface implied a nonuniform distribution of the Type III hexokinase and may represent preferential association with nuclear pore structures. A study of technical factors involved in optimizing the reflection image was conducted. We demonstrate that both the choice of objective and the thickness of the mounting medium are critical to successful imaging, and we describe a simple test for assessing the suitability of objectives in any system.