Immunocytochemical methodology

It is advisable to nrinimize the amoimts of primary antibodies used to reduce cost. Staining sections flat on an enclosed humid incubating tray will necessitate 100-200 jJ of reagent per sUde. Alternatively a qrstem such as the Sequenza Immrmostaining System (Shandon Scientific Ltd.) can be irsed, in which a disposable Coveiplate is closely attached to the slide forming a well over the entire area of the slide with a volume of approximately 100 jJ, into which all necessary solutions can be introduced. The system is efficient and. simple to use. Autonrated immrmostainers are available from many companies in the field. 

To produce consistent results it is necessary to pay attention to all detaUs of the method(s) chosen, in particular timings, composition and pH of solutions, and storage conditions of antibodies. Itnmrmoc54 ochemistry is a craft as well as a science time spent observing an experienced worker is well spent, and practise wiU make perfect 

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Labeling of probes for in situ hybridization relies on the incorporation of either a radioisotopic dNTP (e.g., dCTP), or of a nonisotopic molecule, such as biotin-7-dAlT or biotin-11-dUTP, by either nick translation or random priming. The site (s) of hybridization can then be seen using autoradiography with isotopic probes, or immunocytochemically if biotin is incorporated into the probe DNA. It is with the latter form of in situ hybridization methodology that this chapter is concerned. 

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