Simple immunohistochemical staining method using large sized gold colloid conjugated secondary antibody

To test the feasibility of semiquantitative immunohistochemical staining (IHC) with large sized gold conjugated secondary antibody (gold-2°Ab), we used beads covered with a known amount of primary antibody and a secondary antibody conjugated with gold colloid particles (20 and 40 nm in diameter), and we compared the results to those obtained by enzyme IHC. Beads coated with 6 graded amounts of mouse IgG molecules showed 6 levels of color intensity. The graded color intensities could readily be distinguished. The color developed as soon as we added gold-2°Ab, and the intensities were stable for 1 wk. Enzyme IHC using identical beads showed dregs of pigment after incubation in DAB for 5 min. The large sized gold-2°Ab showed strong signals on cell surfaces; application of the large sized gold-2°Ab to paraffin-embedded tissue sections was also feasible. The color was bright red and was easier to differentiate from hemosiderin pigment than the color developed by enzyme IHC. In conclusion, gold IHC with large sized gold-2°Ab is superior to enzyme IHC for quantification of antigens via IHC. Gold IHC is especially recommended for tissues with many macrophages, such as bone marrow and spleen.