Multiple staining

Van der Loos CM. Immunoenzyme Multiple Staining Methods. Oxford, UK Bios Scientific Publishers, 1999. 

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. 

Finally, it is important to be aware of the fact that visualization systems with dual recognition such as the EnVision + Dual Link System do not discriminate between species, and thus are only suitable for multiple staining when using the sequential method. Visualization kits with amplification layers that are not well specified should be avoided since possible cross-reactivity cannot be predicted. 

Automated Image Acquisition and Analysis in Multiple Staining... 

Detection of different epitopes in the same preparation Multiple staining methods to distinguish different epitopes offer important advantages. In particular, the availability of monoclonal antibodies is likely to result in the use of this method on an increasing scale. 

Multiple staining can be achieved by two, fundamentally different approaches (i) the same enzyme is used for both epitopes but with different substrates, yielding distinctly colored reaction products and, (ii) two unrelated enzymes are used to detect the two epitopes. 

Lanciego JL, Goede PH, Witter MP, Wouterlood FG (1997) Use of peroxidase substrate vector VIP for multiple staining light microscopy. J Neurosci Methods 74 1-7... 

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