Silver enhancement-of polymerised diaminobenzidine: Increased sensitivity for immunoperoxidase staining

Unambiguous identification of lymphocytes is sometimes difficult because of weak immunostaining of the cell membrane immunoglobulins. A simple method of intensifying the diaminobenzidine (DAB) peroxidase reaction was therefore devised. Paraffin wax sections of formalin fixed tonsils and lymphomas were digested with trypsin and immunostained for K and A light immunoglobulin chains and CD3 antigen by various peroxidase linked detection systems. After reaction with hydrogen peroxide and DAB the sections were immersed in methenamine silver solution at 60°C for three to seven minutes. The light brown stain on the cell membranes of the mantle zone lymphocytes became dark brown and the stronger stain of the plasma cells became black. Mantle zone B lymphocytes and CD3 positive T lymphocytes were precisely outlined even at low magnification and the lymphomas were easily classified as monoclonal or polyclonal. At high magnification, staining was clearer than with the immunogold-silver stain. Cryostat and paraffin wax sections of other tissues immunostained for various antigens showed similar intensification. Silver methenamine provides an easy means of increasing the sensitivity and visual impact of an immunoperoxidase/ DAB reaction in any preparation. Department of Histopathology, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN C S Peacock S Van Noorden Department of Histopathology, St Bartholomew's Hospital, London I W Thompson Correspondence to: Miss S Van Noorden Accepted for publication 25 April 1991 Since the establishment of the peroxidase anti-peroxidase (PAP) method' and the avidin-biotin complex (ABC) method2 various other ways of increasing the sensitivity of immunostaining methods have been proposed, with the aim of identifying very small quantities of antigen and of increasing the visual impact of the reaction. Among the earlier suggestions were the addition of imidazole' or heavy metal salts such as cobalt chloride or nickel sulphate4 to the diaminobenzidine (DAB) peroxidase development medium to darken the colour of the normal brown end-product. A different tack was taken by Holgate et al' with the introduction of the immunogold-silver staining (IGSS) method for light microscopy, the scarcely visible red colour of the colloidal gold marker being made intensely black by deposition of silver from a silver lactate solution. This additional intensification allowed the depiction of the surface membrane immunoglobulins of lymphocytes in paraffin wax sections to a much greater extent than ordinary immunoperoxidase techniques.6 Subsequent work by this group resulted in modification of the fixative to give even better localisation of lymphocyte surface antigens.78 Even without this fixative reasonable IGSS staining can be achieved, certainly better than that provided by a standard three-layer immunoperoxidase method, but an inherent problem of the IGSS method is a fine grained non-specific deposit of silver over the section. Scopsi and Larsson carried out an in vitro survey of the sensitivity of available immunoperoxidase methods, including the addition of imidazole and heavy metals to the DAB incubating solution and post-silvering of the reaction product.9 Their results indicated that one of the most sensitive methods was to intensify the oxidised DAB product with silver from a methenamine silver solution.'0 Because this solution is part of a commonly used method of staining basement membranes and fungi,"12 it is readily available in most histology laboratories, and we have found it to be a convenient and valuable way of increasing the sensitivity of three-layer immunoperoxidase staining to the level achieved by IGSS. Methods The methenamine silver intensification can be used on any immunoperoxidase preparation after the peroxidase/H202/DAB reaction has been carried out to give a brown deposit. The preparations are washed in running tap water, then thoroughly rinsed in distilled water, and placed in preheated methenamine silver solution at 60°C for three to five minutes or, occasionally, longer. The intensification can be carried out at room temperature but proceeds at a much slower rate. Sections can be stored indefinitely in distilled water before the silver intensification is carried out, and preparations previously stained weakly by immunoperoxidase and DAB can be retrospectively intensified. In this case it is best to remove haematoxylin from counterstained nuclei with acid alcohol before the silver intensification is carried out. The composition of the stock methenamine silver solution is as follows: 0-125% silver nitrate in 1-5% hexamine; this solution can be stored at 4°C. Just before use, 2 ml of 5% sodium tetraborate is added to 50 ml of the stock silver solution, giving a pH of 8-0. The 756 group.bmj.com on June 19, 2017 Published by http://jcp.bmj.com/ Downloaded from