ADVANTAGES OF BACKSCATTER ELECTRON IMAGING SCANNING ELECTRON-MICROSCOPY FOR INTRACELLULAR-LOCALIZATION OF CARDIAC ANALYTES BY GOLD CONJUGATED ANTIBODY

Myoglobin and myosin light chain 1 (MLC1) are intracellular human card iac marker proteins which are released as a consequence of ischemia. H uman cardiomyocytes were isolated from fresh biopsies and also maintai ned for several passages in cell culture. The cardiomyocytes were fixe d in 100% methanol at -20 degrees C, and labeled. The immunolocalizati on of intracellular antigen by fluorescence conjugated imaging was com pared with scanning electron microscopy (SEM) backscatter electron (BS E) imaging of gold conjugated antibody. Ultraviolet light microscopy s howed the intracellular distribution of both proteins to be mainly in the nuclear envelope, the cytoplasm immediately surrounding the nucleu s and along portions of the cell membrane. To confirm this observed di stribution of myoglobin and MLC1, labeling was repeated with antimyogl obin and anti-MLC1 monoclonal antibodies conjugated to colloidal gold particles. The advantage of colloidal gold labeling is that the intrac ellular antigen-antibody complexes may be more precisely located becau se of the significant improvement in resolution provided by BSE imagin g in the SEM. BSE imaging confirmed the presence and subsarcolemma loc alization of myoglobin in cardiomyocytes directly isolated from fresh biopsies. The distribution of colloidal gold-conjugated antibodies did not coincide with the intracellular distribution of the two proteins in the cardiomyocytes grown in cell culture as indicated by immunofluo rescence. A relatively random, intracellular gold particle distributio n was confirmed by x-ray microanalysis. BSE imaging resulted in consis tent auto-backscatter labeling patterns very similar to the labeling p atterns obtained with immunofluorescent labeling. X-ray microanalysis confirmed that these auto-backscatter labeling patterns were formed by concentrations of intracellular phosphate. Sodium dodecyl sulfate-pol y acrylamide gel eletrophoresis (SDS-PAGE) and subsequent Western blot ting indicated that myoglobin and MLC1 were no longer present in detec table quantities in these cells after several passages. Polymerase cha in reaction (PCR) amplification of mRNA for human myoglobin and cardia c MLC1 confirmed the absence of their transcripts. Electrophoretic ana lysis of proteins in cardiomyocytes grown in cell culture confirmed an increasing presence of alkaline phosphatase. Staining of this enzyme with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium sh owed that alkaline phosphatase was distributed in the same intracellul ar pattern as the fluorescence conjugated antibody and the phosphate a uto-backscatter. These results indicate that high-resolution backscatt er SEM imaging may be used as necessary control to confirm fluorescenc e light microscope intracellular labeling of antigens.