Immunofluorescent analysis of creatine kinase in cultured astrocytes by conventional and confocal microscopy: a nuclear localization.
The subcellular localization of creatine kinase (CK) was examined in primary cultures of astrocytes with immunofluorescent labeling methods and detection by both standard fluorescence microscopy and confocal laser-scanning microscopy. With conventional microscopy, the pattern of CK staining was uniform throughout the cell cytoplasm and appeared to stain the nuclear region intensely. Staining of CK in the nuclear region co-localized with the DNA-specific Hoechst nuclear stain. CK produced a diffuse cytoplasmic staining pattern that was different from the staining pattern produced by the cytoskeletal proteins glial fibrillary acidic protein and tubulin, both of which showed a filamentous cytoskeletal network that excluded the nucleus. To examine the structural details of CK in the nuclear region, serial optical sections were taken through the cell monolayer with a confocal microscope. The cells were immunostained for CK, and the CK-staining pattern was compared with the staining pattern produced by propidium iodide, which is specific for DNA in RNase-treated samples and stains total nucleic acid in untreated samples. CK staining was present within the nucleus in each section taken through the monolayer. The nucleolus did not stain for CK. The pattern of CK staining in the nucleus (and cytoplasm) was distinctly different from the staining pattern of either DNA or total nucleic acid. Nuclear CK appeared to have a granular, particulate pattern, which is suggestive of a nucleoplasmic distribution.
D000818:Animals; D001253:Astrocytes / Q000201:enzymology* / Q000648:ultrastructure; D006690:Bisbenzimidazole; D002467:Cell Nucleus / Q000201:enzymology*; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D008853:Microscopy / Q000379:methods; D008856:Microscopy, Fluorescence; D051381:Rats
D003402:Creatine Kinase; D006690:Bisbenzimidazole