A one-day double-labelling 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 sensitivity, specificity, the use of hazardous radioactive equipment and time consumption in immunohistochemical labelling and double labelling of in situ hybridization of tissue specimens. It consists of a two-step protocol in which digoxigenin-uridine triphosphate (UTP) labelled riboprobes in the in situ hybridization step are visualized by the immunogold-silver staining method, and double labelling of tissue antigens is achieved by the application of an alkaline phosphatase-anti-alkaline phosphatase staining step. We tested this protocol using snap-frozen tissue sections of synovial tissue from patients with rheumatoid arthritis. The target mRNA was detected by perforin or cathepsin D riboprobes, the double labelling was performed using anti-collagen type IV and alpha-smooth muscle actin antibodies. It is concluded that, in comparison 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 for double labelling of in situ hybridization products and tissue antigens.
Müller-Ladner
U
U
Department of Medicine, University of Alabama at Birmingham 35294-0006, USA.
Kriegsmann
J
J
Gay
R E
RE
Gay
S
S
D000469:Alkaline Phosphatase / Q000276:immunology; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology*; D002403:Cathepsins / Q000032:analysis / Q000235:genetics; D003094:Collagen / Q000032:analysis; D004076:Digoxigenin; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics; D054353:Perforin; D010510:Periodontal Diseases / Q000473:pathology; D052899:Pore Forming Cytotoxic Proteins; D015347:RNA Probes; D016622:Silver Staining; D013583:Synovial Membrane / Q000473:pathology / Q000648:ultrastructure
D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D015347:RNA Probes; D054353:Perforin; D003094:Collagen; D000469:Alkaline Phosphatase; D002403:Cathepsins; D004076:Digoxigenin