Practical suggestions for successful immunoenzyme double-staining experiments

A new method for demonstrating antigens in paraffin sections of formol sublimate-fixed tissue is described that utilizes an "indirect" immunohistological technique employing immunoglobulin adsorbed to colloidal gold as the secondary antiserum. The gold particles introduced to antigenic sites are revealed by a silver precipitation reaction. This technique, the immunogold-silver staining method, is of much enhanced sensitivity (up to 200-fold) as compared with standard immunoperoxidase and immunogold staining methods. The results have been confirmed in a study of immunoglobulins in reactive human tonsil. The use of this new method for double immunolabeling is also described.

Three metallic ions, NiCl2, CoCl2, and CuSO4, were found to modify the color of the normally brown diaminobenzidine (DAB) reaction. The colors ranged from purplish blue (NiCl2), dark blue/bluish black (CoCl2), to greyish blue (CuSO4). We have found that the CoCl2 + DAB is the ion of choice because: 1) it yields a distinct dark blue color that is easily distinguishable from brown DAB; 2) the blue reaction product is very stable throughout the entire staining procedure; and 3) background staining is minimal. These findings can be applied to the double staining technique of two different antigens in the same section. Among three staining procedures discussed, the avidin-biotin peroxidase complex (Co-DAB)-peroxidase-antiperoxidase (DAB) technique produced the best results because: 1) no antibody elution was needed following the avidin-biotin-peroxidase complex procedure when the CoCl2-DAB modification was used; and 2) no background staining occurred.