Immunogold labeling of human leukocytes for scanning electron microscopy and light microscopy: quantitative aspects of the methodology

Abstract

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.