Simultaneous Immunoenzymatic Double Staining

A collection of useful protocols for the simultaneous immunoenzyme staining of two or more tissue antigens is available on the websites http //www.protocol-online.org/prot/Immunology/ http //www.vectorlabs.com/Protocols/MLB.pdf and http //www.ihcworld.com/. The protocols given in this chapter are practiced in the authors laboratory. These methods fall into two main categories (a) simultaneous immunoenzymatic double staining, and (b) sequential immunoenzymatic double/ multiple staining. 

Buchwalow and W. Bocker, Immunohistochemistry Basics and Methods, DOI 10.1007/978 3 642 04609 4 7, Springer Verlag Berlin Heidelberg 2010 

Basic protocol for simultaneous immunoenzymatic double staining using primary antibodies of two different species or two different IgG isotypes 

Deparaffinize and rehydrate tissue sections. Rinse in distilled water for 5 min. 

Antigen retrieval place sections in a Coplin jar with antigen retrieval solution of choice (e.g., 10 mM citrate acid, pH 6) and heat at 90 110°C (depending on tissue) in a microwave, steamer, domestic pressure cooker or autoclave (see Sect. 6.1.1). 

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