The application of microwave techniques in multiple immunostaining and in situ hybridization

We describe simple, rapid, reliable and sensitive microwave-based methods for both multiple immunostaining and in situ hybridization. This technique involves treating sections with a 10 min period of microwave oven heating. The ability of microwave oven heating to retrieve cytoplasmic and nuclear antigens, block antibody cross-reactivity and inactivate peroxidase and alkaline phosphatase enzymes has resulted in the development of a simple and generally applicable method of multiple immunostaining in which two or more antigens can be detected within the same section using primary antibodies raised in the same species combined with a sensitive three layer staining protocol involving peroxidase anti-peroxidase (PAP) and alkaline phosphatase anti-alkaline phosphatase (APAAP) complexes. The application of microwave oven heating as a pretreatment of cryostat sections prior to in situ hybridization using digoxigenin-labelled cRNA and cDNA probes resulted in improved probe access to target nucleic acid sequences, stronger hybridization signals, reduced hybridization times, maintenance of excellent tissue morphology and outstanding reliability. Thus, the application of microwave oven heating has overcome many of the difficulties associated with current protocols used in these two techniques. These new methodologies have general applicability and will be of significant benefit to both research scientists and clinical pathologists alike.