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 cardiac marker proteins which are released as a consequence of ischemia. Human cardiomyocytes were isolated from fresh biopsies and also maintained for several passages in cell culture. The cardiomyocytes were fixed in 100% methanol at -20 degrees C, and labeled. The immunolocalization of intracellular antigen by fluorescence conjugated imaging was compared with scanning electron microscopy (SEM) backscatter electron (BSE) imaging of gold conjugated antibody. Ultra-violet light microscopy showed the intracellular distribution of both proteins to be mainly in the nuclear envelope, the cytoplasm immediately surrounding the nucleus and along portions of the cell membrane. To confirm this observed distribution of myoglobin and MLC1, labeling was repeated with antimyoglobin and anti-MLC1 monoclonal antibodies conjugated to colloidal gold particles. The advantage of colloidal gold labeling is that the intracellular antigen-antibody complexes may be more precisely located because of the significant improvement in resolution provided by BSE imaging in the SEM. BSE imaging confirmed the presence and subsarcolemma localization 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 immunofluorescence. A relatively random, intracellular gold particle distribution was confirmed by x-ray microanalysis. BSE imaging resulted in consistent auto-backscatter labeling patterns very similar to the labeling patterns obtained with immunofluorescent labeling. X-ray microanalysis confirmed that these auto-backscatter labeling patterns were formed by concentrations of intracellular phosphate. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting indicated that myoglobin and MLC1 were no longer present in detectable quantities in these cells after several passages. Polymerase chain reaction (PCR) amplification of mRNA for human myoglobin and cardiac MLC1 confirmed the absence of their transcripts. Electrophoretic analysis 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 showed that alkaline phosphatase was distributed in the same intracellular pattern as the fluorescence conjugated anti-body and the phosphatase auto-backscatter. These results indicate that high-resolution backscatter SEM imaging may be used as necessary control to confirm fluorescence light microscope intracellular labeling of antigens.
Lea
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Spectral Diagnostics Inc., Toronto, Ontario, Canada.
Lee
L M
LM
Shi
Q W
QW
Takahashi
M
M
Youn
W
W
Jackowski
G
G
D000469:Alkaline Phosphatase / Q000032:analysis; D001483:Base Sequence; D001706:Biopsy; D015153:Blotting, Western; D004577:Electron Probe Microanalysis; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D006352:Heart Ventricles / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000201:enzymology / Q000648:ultrastructure*; D009206:Myocardium / Q000166:cytology*; D016133:Polymerase Chain Reaction; D012542:Scattering, Radiation
D000469:Alkaline Phosphatase; D016650:Fluorescein-5-isothiocyanate