Immunolabeling

Nicolas, M.-T., Morse, D., Bassot, J. M., and Hastings, J. W. (1991). Colocalization of luciferin-binding protein and luciferase to the scintillons of Gonyaulax polyedra revealed by double immunolabeling after fast-freeze fixation. Protoplasma 160 159-166. 

Helfert, RH, Juiz, JM, Bledsoe, SC, Bonneau, JM, Wenthold, RJ and Altschuler, RA (1992) Patterns of glutamate, glycine and GABA immunolabelling in four synaptic terminal classes in the lateral superior olive of the guinea pig. J. Comp. Neurol. 323 305-325. 

Fig. 11.2. A donor dequenching experiment that demonstrates that extracellular domains of APP and LRP closely associated with one another. H4 cells were transfected with APP770 and LRP and immunolabeled with Fluorescein (A) and Cy3 (B), respectively. When the Cy3 is photobleached in the area marked by the white rectangle, the signal from fluorescein increases (C), while the signal from Cy3 disappears (D). The increase in donor fluorescence is calculated to be 51%, which shows that the ectodomains of APP770 and LRP, are in close proximity and therefore likely to be interacting. This result has implications for the field of Alzheimer s disease research as it helps elucidate the nature of APP processing into amyloid-/ [50],...

The two most established methods of labeling a molecule or pair of molecules to make a FRET measurement are immunolabeling and fusion to genetically encoded FPs like GFP. Although these are well established techniques, they have certain drawbacks and so novel sensors like QDs and labeling techniques, like cysteine-reactive fluorophores continue to be developed and offer great promise for the future. 

Kanitakis, J. and Thivolet, J. (1987). Immunolabeling methods in cutaneous histopathology. Principles and practical applications. Ann. Pathol. 7,79-97. 

Tang X, Falls DL, Li X, et al. Antigen-retrieval procedure for bromodeoxyuridine immunolabeling with concurrent labeling of nuclear DNA and antigens damaged by HC1 pretreatment. J. Neurosci. 2007 27 5837-5844. 

Bonnard, C., Papermasteg D.S., and Kraehenbuhl, J.-P. (1984) The streptavidin-biodn bridge technique Application in light and electron microscope immunocytochemistry. In Immunolabelling for Electron Microscopy, (J.M. Polak, and I.M. Varndell, eds.), pp. 95-111. Elsevier, New York. 

FIGURE 34-5 Induction of endothelial nitric oxide synthase (eNOS) in medial thalamus of thiamine-deficient rats. (A) Increased eNOS mRNA. (B) Increased eNOS immunolabeling of vascular endothelial cells (magnification x200). 

Not detected High levels Dual immunolabeling Hrabovszky et al. 2001... 

Beesley JE. Multiple immunolabeling techniques, mlmmunocytochemistry, APrac-tical Approach (Beesley JE, ed.), Oxford University Press, Oxford, UK, 1993, pp. 103-125. 

After blocking, the initial step for immunolabeling is to incubate grids in blocking solution in which is diluted the primary antibody. Grids can either be floated on the solution or immersed within in it. We use... 

Immunolabel sections (in the case of Sawada and Esaki, nanogold was conjugated to the secondary antibodies and silver enhancement was employed [34].)... 

Kollack-Walker, S. and Newman, S. (1995) Mating and agonistic behavior produce different patterns of Fos immunolabeling in male Syrian hamster brain. Neuroscience 66, 721-36. 

For double immunolabeling with primary antibodies from the same host species, it is not necessary to resort to labeling with monovalent Fab fragments when primary antibodies from the same host species are different isotypes (subclasses) of IgG, for instance such as mouse IgGl and mouse IgG3. In these cases, isotype-specific antibodies may be used to distinguish between the two primary antibodies... 

Brown JK, Pemberton AD, Wright SH, Miller HRP (2004) Primary antibody Fab fragment complexes a flexible alternative to traditional direct and indirect immunolabeling techniques. J Histochem Cytochem 52 1219 1230... 

Fc receptors present on the cell membrane of macrophages, monocytes, granulocytes, lymphocytes, and some other cells may, in theory, non-specifically bind Fc portion of antibodies used for immunolabeling. Typically, blocking endogenous Fc receptors involves using normal serum derived from the same species used to produce the secondary antibody. To block endogenous Fc receptors, incubate sections for 15 30 min with normal serum (2 5% v/v) from the host species of the secondary antibody. 

Figure 7.1 illustrates the application of this technique with double immunolabel-ing of two antigens, Ki67 (Mouse IgG) and Von Willebrand Factor (F8, Rabbit IgG)... 

The protocol for double/multiple immunolabeling using haptenylated primary antibodies is essentially the same as with primary antibodies of different IgG isotypes. These protocols can be easily customized depending on the availability of primary antibodies for your research requirements. For instance, you may have at your disposal a pair of monoclonal antibodies of the same IgG isotype, and only one of them is haptenylated. In this case, you have to carry out the immunostaining in two steps in the first step you visualize the unlabeled first primary antibody with a secondary species-specific antibody, and in the second step you can detect the second primary haptenylated antibody via another secondary antibody directed against the corresponding hapten. Should the hapten be a fluorophore, it can be visualized directly in a fluorescent microscope and you do not need the second step... 

The ratio of primary antibodies to Fab fragments required for the formation of complexes that produce optimal immunolabeling of specific antigen does not appear to vary dramatically with primary antibody specificity or species. Primary antibody to Fab fragment ratios of 1 2 1 4 (weight for weight, based on concentration data supplied by manufacturers) typically produces optimal results with primary mouse monoclonal antibodies. 

Immunohistochemical labeling for electron microscopy is based on the same principles as immunohistochemistry for light microscopy. The differences are that specimen sections must be much thinner (50 100 nm) and the label must be electron-dense. The first electron-dense labels used for immunolabeling at the electron microscope level were ferritin and peroxidase. Peroxidase label can be visualized using DAB reaction product which becomes electron-dense after osmi-cation. With the advent of colloidal gold particles as markers in immunocytochemical... 

---