THE VISUALISATION OF TRACE AMOUNTS OF DIAMINOBENZIDINE (DAB) POLYMER BY A NOVEL GOLD‐SULPHIDE‐SILVER METHOD

A feature of immunoperoxidase techniques which limits their sensitivity is the low optical density of DAB when it is deposited in small amounts. This is true, for example, of glass-slide-adhered semi-thin sections of -L R White embedded tissue where, following immunoperoxidase staining without etching or protease digestion, the DAB reaction product is barely visible even though antigenicity can easily be demonstrated a t the electron microscope level under these conditions (Newman et al, 1982, 1983a). In efforts to improve i t s optical density, metal compounds have been applied to DAB in light microscope immunocytochemistry (Adams 1981). However, the use of metal salts, although altering the colour, does not greatly enhance the optical density of DAB. In addition, the use of DAB to catalyse directly the deposition of silver (Gallyas e t ai 1982) results in only slight improvement (Fig. l a ) . DAB polymer may also be difficult to see with the electron microscope, but in a study exploring the use of metal compounds, we have shown that this is easily corrected using gold chloride, for which the polymer has a strong affinity (Newman et al 1983b). With this information we have therefore developed a technique which greatly intensifies the optical density of DAB and which is based on three separate principles that DAB has a high affinity for metal salts, that the sulphides of insoluble metals catalyse the deposition of silver in the presence of a reducing agent (Timm 1958) and that the powerful light insensitive developing solution of Gallyas e t al can then be used much more effectively, and avoids the need for darkroom conditions. We have demonstrated the value of this technique in 750 nm sections of formalin fixed L R White embedded tissue. After immunoperoxidase staining, the sections were treated with 0.1% gold chloride for 5 minutes, thoroughly washed, and immersed in neutralised sodium sulphide for a further 5 minutes. After again thoroughly rinsing the sections, they were exposed to the silver developing solution of Gallyas e t al (1982) for 60 90 sec, and the reaction stopped by immersing the sections in 1% acetic acid (results illustrated in Figs. l b , 2 & 3). In experiments, when gold chloride was used alone, only slight improvement in optical density of DAB was seen, and when the sulphide step was omitted, the developer was ineffective. This technique differs from that of Holgate et al (1983) in that the tissue under examination needs no special fixation, the method i s simple to use (does not require complex, expensive chemicals and darkroom conditions), and is directly applicable to the widely used immunoperoxidase technique (does not require the production of colloidal gold immunocomplexes). We have shown that it is readily suited to the immunostaining of serial semithin sections of L R White embedded tissue where the analysis of the distribution of multiple antigens in one tissue is an obvious application. We have also used the technique in preliminary studies of the detection of surface antigens of lymphoid cells, and extracellular immunoglobulins in the kidney. We are currently exploring the use of this very sensitive technique in a number of other situations.