Immunocytochemical applications

As noted above, the specificity of the antibody-antigen reaction is critical for obtaining reliable, interpretable results. For this reason, the antibody has to be tested rigorously, and essential controls for antibody specificity should be included in any experimental design. A comprehensive discussion on antibody generation, specificity, and testing for immunocytochemical applications can be found in references (27-29) and, for specific applications, see Chapters 17, 50, and 51. 

Chagnaud JL, Campistron G, Geffard M (1989) Monoclonal antibody directed against glutaraldehyde conjugated glutamate and immunocytochemical applications in the rat brain. Brain Res 481 75-180. 

Campistron G, Buijs RM, Geffard M (1986) Specific antibodies against aspartate and their immunocytochemical application in the rat brain. Brain Res 365 179-184. 

Somogyi P, Hodgson AJ, Chubb IW, Penke B, Erdei A (1985) Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system. J Histochem Cytochem 33 240-248... 

Montone KT, Brigati DJ, Budgeon LR. Anatomic viral detection is automated the application of a robotic molecular pathology system for the detection of DNA viruses in anatomic pathology substrates, using immunocytochemical and nucleic acid hybridization techniques. Yale J. Biol. Med. 1989 62 141-158. 

Weintraub J, Redard M, Wenger D, et al. The application of immunocytochemical techniques to routinely fixed and stained cytologic specimens. Pathol. Res. Pract. 1990 186 658-665. 

The final application of the antibody must be borne in mind when deciding the extent of characterization. Initially, the antibody must be tested to establish whether binding occurs with the immunogen, with and without any carrier molecules used in the immunization. This test should be carried out with reference to the intended application to control for bridge binding. For instance, a reagent intended for use in a capture ELISA should be tested when coated onto the assay solid phase. If the antibody is intended for in vivo immunoneutralization, it should be tested initially in a liquid phase assay. If the final use is to be immunocytochemical, then the testing should be conducted on tissue sections. 

In recent years, the immunocytochemical approach using avidin-biodn technology has experienced an addidonal flurry of acdvity owing to its applicability in pathodiagnosdcs. 

Immunocytochemical methods have found broad application in the clinical, as well as the research setting. Clinically relevant specimens ranging from frozen sections and cell-touch preparations to whole-tissue samples are amenable to analysis see Chapters 9-13). Panels of antibodies have been developed to aid in the differential diagnosis of tumors see Chapter 48), and automated instrumentation has been designed to speed the handling of numerous specimens see Chapter 47). 

The merger of traditional immunocytochemical methods with traditional nucleic acid hybridization techniques has resulted in the development of experimental approaches that serve very effectively for the detection and localization of nucleic acids in biological materials. Furthermore, these in situ methods also may serve for the molecular characterization (e.g., base composition) and quantitation (e.g., copy number) of such nucleic acids. Because of the ubiquitous occurrence of nucleic acids and the sensitivity of the detection methods, the applications are broad and diverse, and often yield information not accessible by other methods. 

Beaty MW, Fetsch P, Wildet AM, et al. Effusion cytology of malignant melanoma. A motphologic and immunocytochemical analysis including application of the MART-1 antibody. Cancer. 1997 81 57-63. 

Consideration of the structure and distribution of cannabinoid receptors is critical to the successful application of immunocytochemical approaches to the identification of cannabinoid receptors in cells and tissues. CBi and CB2 receptors exhibit greatest type specificity at their extracellular amine-terminal domains. Thus, experiments designed for identification of cannabinoid type-specific receptor expression at the protein level should entail use of affinity-purified antibodies directed against the amine-terminal domains. Use of such antibodies is also critical for the identification of cell surface expression of cannabinoid receptors. On the other hand, if assessment of expression of fully processed receptor protein is required, then antibodies directed against carboxy-terminal domains of the CBi or CB2 should be considered. Immunocytochemical methods in which such antibody preparations are considered should include a limited cell/tissue solubilization so as to allow for adequate antibody penetration. A list of commercially available antibodies to domains of CBi and CB2 receptors is included in Table 1. In addition, various laboratories have applied these commercially available antibodies (or have developed their own antibodies) to the identification of cannabinoid receptors at the fluorescence, light, and electron microscopy levels. Table 2 lists references in which immunocytochemical approaches have been utilized for the identification of cannabinoid receptors in a variety of tissues and cell types. 

The objective of the present chapter is to provide a few standard protocols for immunocytochemical detection of neuropeptides, including double and triple labeling techniques that utilize difierent primary antisera/antibodies. It is very important to remember that ICC protocols have been modified in almost every laboratory that employs the technique, and that almost every antiserum or tissue requires specific modifications. Thus, it is wise to consult the original papers describing initial characterization of the antiserum and its application to specific tissues and animal species. For commercially available antisera, standard protocols are commonly supplied. The protocols presented here should provide a starting point for peptide ICC, and we suggest points at which modifications can be made. 

The application of immunocytochemistry to ammo acid transmitter systems m the central nervous system has provided valuable information on the cellular and subcellular locations of these transmitter systems It is particularly suited to the detection of the GABA system, and this topic forms the focal point of the chapter on immunocytochemical techniques. Although the im-munocytochemical approach is extremely valuable m specific situations, the method is of limited applicability m the amino acid field, only the GABA and taurine systems having been mapped by these procedures. 

Demski and Northcutt (1983) did find, however, that application of horseradish peroxidase to the nasal epithelium resulted in transport of this enz3mie to the TN ganglion and its axon terminals in the telencephalon, suggesting proximity of TN dendrites and olfactory epithelium. Indeed, immunocytochemical visualization of the TN of goldfish revealed processes extending from the ganglion cells along the branches of the olfactory nerves to the base of the olfactory lamellae, but no processes were observed in the sensory epithelium itself (Kyle, unpublished results). Clearly, a role for the TN in pheromone detection has yet to be established. 

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