A ONE-DAY DOUBLE-LABELING TECHNIQUE FOR TISSUE SPECIMENS - IMMUNOGOLD-SILVER STAINING FOR IN-SITU HYBRIDIZATION COMBINED WITH ALKALINE PHOSPHATASE-ANTI-ALKALINE PHOSPHATASE (APAAP) IMMUNOHISTOCHEMISTRY FOR ANTIGENS

An improved technique is described that addresses the problems of sens itivity, specificity, the use of hazardous radioactive equipment and t ime consumption in immunohistochemical labelling and double labelling of in situ hybridization of tissue specimens. It consists of a two-ste p protocol in which digoxigenin-uridine triphosphate (UTP) labelled ri boprobes in the in situ hybridization step are visualized by the immun ogold-silver staining method, and double labelling of tissue antigens is achieved by the application of an alkaline phosphatase-anti-alkalin e phosphatase staining step. We tested this protocol using snap-frozen tissue sections of synovial tissue from patients with rheumatoid arth ritis. The target mRNA was detected by perforin or cathepsin D ribopro bes, the double labelling was performed using anti-collagen type IV an d alpha-smooth muscle actin antibodies. It is concluded that, in compa rison with an established three- to four-day double-labelling protocol used in many laboratories, this one-day combination is currently the most rapid assay of reliable quality fur double labelling of in situ h ybridization products and tissue antigens.