Immunocytochemistry species

Pirici D, Mogoanta L, Kumar Singh S, Pirici I, Margaritescu C, Simionescu C, Stanescu R (2009) Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtype. J Histochem Cytochem 57(6) 567 575 Polak JM, Van Noorden S (1997) Introduction to immunocytochemistry. BIOS Scientific, Oxford... 

Immunocytochemistry uses antibodies against IgGs. Antibodies or IgG molecules are generated to other IgG molecules by injecting purified IgG molecules from one species into another species. In the case of mouse IgG injected into rabbit, it will produce rabbit anti-mouse IgG antibodies. Antibodies made against an IgG will only bind to the constant region or Fab region of the IgG. 

Terminology is important in describing the source and specificity of antibodies used in immunocytochemistry. The species used to generate antibodies are used to differentiate antibodies. An antibody generated in rabbit to the protein mbulin would be a rabbit anti-tubulin antibody. With both mouse and rabbit being used to make monoclonal antibodies, the species of the animal generating the monoclonal antibody must be stated, and not simply monoclonal to mean antibodies produced in mouse. To identify an antibody, use the species of animal where the antibody was generated and not the term monoclonal. 

Fig. 5.2 Examples of nonspecific antibody binding. During immunocytochemistry incubations nonspecific binding occurs. The correct antigen will bind the primary antibody correctly. Charged groups will bind to any protein or antibody such as the 1° antibody. The Fc receptor will bind the first antibody that comes in contact here with the 1° antibody. Any endogenous antibodies are likely to bind only to secondary antibodies made against the species of the animal...

Fig. 7.2 Indirect immunocytochemistry. An unlabeled 1° antibody binds to the antigen and a labeled 2° antibody binds to the 1° antibody. The labeled 2° antibody is made in a different species of animals against the IgG species of the 1° antibody. A variety of labels can be used from fluorescence to enzymes...

A major advantage to the indirect method is that each labeled 2° will attach to all 1° antibodies from one species (e.g., goat anti-rabbit IgG labeled with 488 flu-orophore works for all rabbit 1° antibodies). This is the method of choice today because so many different antibodies are used in biomedical research. Indirect immunocytochemistry detects proteins in cells with the high detection sensitivity, good flexibility of reagents, and the fewest steps. 

Steps in a two 1° antibody different species indirect immunocytochemistry experiment. 

When an experiment uses multiple 1° antibodies derived from the same species, perform the incubations for the first 1° and 2° antibodies, block the remaining antibody sites, and then perform the incubations for the second 1° and 2° antibodies. This procedure consists of a series of two single U antibody indirect immunocytochemistry experiments with extensive blocking between them. The key element is the blocking steps between. In this example, cultures are incubated with the first 1° antibody set, mouse anti-Ag A and 2° antibody goat anti-mouse labeled with 488 fluorophore (Fig. 12.1a), followed by steps that block the remaining antibody sites (Fig. 12.1b, c). The incubations with the second 1° antibody set are with mouse anti-Ag B antibody and 2° antibody goat anti-mouse... 

Houser CR, Barber RP, Crawford CD, Matthews DA, Phelps PE, Salvaterra PM, Vaughn JE (1984) Species-specific second antibodies reduce spurious staining in immunocytochemistry. J Histochem Cytochem 32 395 02. 

Secondary (2°) antibody - a species-specific antibody that binds to the constant end of other antibodies in immunocytochemistry frequently, it is labeled with a marker seen in the microscope. 

Localization of CCAP in crustaceans and various insect species has been studied in detail with the aid of immunocytochemistry [73, 167]. The distribution, for example, in neurons of brain, midgut and ventral nerve cord of insects suggests multiple and diverse functions, some of those are surely neuromodulatory (immunopositive intemeurons which have processes into brain neuropil) but others are neurohormonal roles... 

Neuropeptides constitute the largest and most diverse class of signaling substances known in metazoans. Over the last 20 yr it has become apparent that neuropeptides have important roles as neurohormones, neuromodulators, cytokines, morphogenetic factors, and possibly in some cases, as true neurotransmitters. Each neuropeptide may even be multifimctional and exist in several isoforms in a given animal species. In the search for functions of neuropeptides, it has been critical to be able to localize sites of synthesis and release. Immunocytochemistry (ICC) has been instrumental in the accurate mapping of the cellular and subcellular distribution of neuropeptides in tissue. Other immunological assays, such as radioimmunoassay (RIA) and immxmo-enzymatic assay (ELISA) provide powerful complements for quantification of neuropeptides. Several important discoveries related to neuropeptides have relied on ICC, for example Different neuropeptides have very specific distributions in small populations of neurons (1—3), neuropeptides are commonly colocalized with low-mol-wt neurotransmitters or other neuropeptides (4), the chemical diversity of neurons is far greater than previously suspected (2,3), and neuropeptide synthesis and release can be episodic (5). 

NADPH diaphorase histochemistry is now the most widely used method for revealing the distribution of NOS in the nervous system. Using this approach, the anatomy of putative NO-producing neurons has been described in the nervous systems of several mammalian species, a variety of other vertebrate species, and invertebrates (7—11). A major advantage of the NADPH diaphorase method over such alternatives as NOS immunocytochemistry and in situ hybridization is that the only specific reagents required are NBT and NADPH, both of which can be easily purchased Moreover, to date anti-NOS antibodies have been raised only to forms of the enzyme obtained from mammals and... 

This book, from its initial conception, had obviously to be limited in the choice of subjects, but we believe it represents a valuable and readily reproducible collection of established and emerging techniques. Such a collection is preceded by a general introductory chapter (Chap. 1) that recalls the history of immunocytochemistry, its basic principles, and its application to the study of neuronal complexity. The methods presented include immunocytochemical localization at light and electronic levels, biochemical characterization, and functional analysis in vivo or ex vivo by novel types of microscopy, as well as protocols for development and production of genetic probes. Although this book is primarily devoted to approaches for analysis of the mammalian brain, a few nonmammalian species are also taken into consideration to demonstrate the importance of alternative animal models in a more comprehensive analysis of central and peripheral neurons. 

---