Immunogold labeling of human leukocytes for scanning electron microscopy and light microscopy: quantitative aspects of the methodology.
When cell surface antigens are labeled with the colloidal gold marker, back-scattered electron images (BEI) reveal all the gold particles and, therefore, permit total counts. Secondary electron images (SEI) show only a small percentage of the gold particles and are inadequate for quantitative evaluation. For determination of the cellular labeling index, a time-consuming method implies the screening of 100 cells by scanning electron microscopy, at a magnification of approximately 12,000 to 15,000x, with continuous SE/BE shifts. A much more efficient method is to transfer the SEM sample or its equivalent under the light microscope and to count the total number of gold labeled cells in the epi-polarization mode. The total cell count can be evaluated under UV light, taking advantage of the autofluorescence of the glutaraldehyde fixed cells.
D000954:Antigens, Surface / Q000032:analysis*; D002462:Cell Membrane / Q000648:ultrastructure; D006046:Gold; D006801:Humans; D007202:Indicators and Reagents; D007962:Leukocytes / Q000166:cytology* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning / Q000295:instrumentation / Q000379:methods
D000954:Antigens, Surface; D007202:Indicators and Reagents; D006046:Gold