Unlabeled antibody methods in electron microscopy: a comparison of single and multistep procedures using colloidal...

A comparative study of five unlabeled antibody methods was conducted on the electron microscopic level using bridging techniques and colloidal gold. The study was based on the principles of the single-step colloidal gold (GLAD) method (Larsson L: Nature 282:743, 1979) and the multistep single- and double-bridge techniques used in postembedding immunoperoxidase procedures (PAP) (Sternberger LA: Immunocytochemistry, 2nd ed. Wiley, New York, 1979). Using medullary thyroid carcinoma and the same lot of primary antiserum (goat anti-calcitonin) for each procedure, it was shown that adequate localization of calcitonin with the single-step GLAD method was attainable only at dilutions of 1:100 or lower. The single-bridge technique using goat anti-calcitonin, sheep anti-goat immunoglobulin (Ig)G, and goat anti-calcitonin and antigen-coated gold, respectively, worked well at dilutions of up to 1:5000 but not at dilutions of 1:10,000, while single- and double-bridging techniques utilizing goat anti-calcitonin, sheep (Sh) anti-goat IgG, and sheep anti-goat IgG-coated gold produced good localization at a 1:10,000 dilution of primary antiserum. A two-step method using goat anti-calcitonin and sheep anti-goat IgG-coated gold, respectively, appeared to be the most sensitive technique, with adequate antigen localization occurring at a dilution of 1:25,000. While in our hands the two-step method appeared superior in sensitivity to the single-bridge IgG-coated gold technique, each method has its own advantages depending on the individual needs of the researcher.