The determination of comparable labeling densities in quantitative immunoelectron microscopic double labeling studies

In quantitative ultrastructural studies using colloidal gold immunocytochemical techniques, labeling intensities vary according to the size of the probe used. Using postembedded indirect two-sided double labeling and single labeling protocols, the labeling characteristics of four antigens were studied using two probe sizes commonly used in double labeling studies. It was determined that the labeling intensity variation resulting from the use of different probe sizes was unpredictable after correcting for the increased probe size alone. It was possible, however, to obtain comparable labeling densities by first determining the labeling intensities for each probe size with its antigen in single label studies on serial sections and using the same procedure as the double labeling studies. A probe size correction factor for each antigen was calculated from these data. This factor was used to obtain comparable measurements of the relative abundance of each label.