Increased sensitivity in immunocytochemistry. Effects of double application of antibodies and of silver intensification on immunogold and peroxidase-antiperoxidase staining techniques

Abstract

Sensitivity and detection efficiency of immunocytochemical methods were tested on cytochemical models and tissue material, respectively. Use of silver intensification procedures revealed that staining with immunogold reagents could be rendered equally or even more sensitive than the standard peroxidase-antiperoxidase (PAP) method. Further increases in sensitivity with both methods could be obtained by double application of the primary antiserum. Combined use of the immunogold techniques and the PAP method with development in diaminobenzidine and subsequent silver intensification resulted in the most sensitive procedures. The procedures were applied to a wide variety of tissue preparations, including whole mount preparations of the external longitudinal muscle layer of the gut wall and were found not to produce any unspecific staining in any tissue tested. Use of immunogold-silver and, particularly of the combined immunogold-silver-PAP methods may be valuable for analyzing tissues and tumours containing small amounts of antigen, for testing the quality of immunogold staining procedures intended for ultrastructural studies and for electroblotting techniques.