Antibodies microscopy

Roberts C J, Williams P M, Davies J, Dawkes A C, Sefton J, Edwards J C, Haymes A G, Bestwick C, Davies M C and Tendler S J B 1995 Real-space differentiation of IgG and IgM antibodies deposited on microtiter wells by scanning force microscopy Langmuir 1822... 

Hirokawa, N., Pfister, K.K., Yorifuji, H., Wagner, M.C., Brady, S.T., Bloom, G.S. (1989). Sub-molecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration. Cell 56, 867-878. 

Keywords. Antibodies, Biosensors, Non-covalent bond. Atomic force microscopy, Supramolecular chemistry... 

Fig. 10. Intercellular junction zones of carrot cells grown in suspension have been observed in electron microscopy after immunogold labeling with the 2F4 antibody, (a) no treatment of the sections prior to labeling the gold particles are restricted to the center of the junction zones (b) enzymatic (pectin methyl esterase) deesterification of the E.M. grids before labeling the deesterified pectins present in the primary walls now bind the probe. Scale bars = 1 pm.

There are very many papers in the literature that address some aspect of gold nanospheres. In particular, their plasmon response (see Section 7.3.1.1) has been well studied, as has their agglomeration [50-52] and the manner in which they can be assembled into highly ordered colloidal crystals [50, 53, 54]. The latter are interesting and will be further discussed in Section 7.3.8.2. Conjugation of gold nanospheres with proteins and antibodies, for use as a stain in microscopy [55] or possibly, in medical applications [23], is another rich field. 

Figure 6 Detail of the interface space (IN) between the fungal wall (U) of Glomuf versiforvte and the host membrane of leek (PL), as seen by electron microscopy. Xylo-glucan molecules are revealed by using a specific antibody and colloidal gold granules. P, host cell F, fungus, X 30,000.

The identity of TES-32 and CTL-1 was confirmed by polyclonal antibodies to recombinant CTL-1, which bound to native TES-32, and by monoclonal antibody Tcn-3, raised to native TES-32 (Maizels et al, 1987), which specifically recognized recombinant CTL-1. The CTL-1 sequence also contained three sites for Afglycosylation, which had previously been shown to be present on TES-32 (Page and Maizels, 1992). Both Tcn-3 and polyclonal antibody to the recombinant CTL-1 protein localize to the cuticle of the infective larvae by immunoelectron microscopy (Fig. 12.2). 

Like the palladium(II) complexes, the platinum(II) porphyrins show appreciable phosphorescence even in aqueous media at room temperature in one study,169 singlet oxygen quantum yields ranged from 0.1 to 0.9 and were strongly influenced by dimerization/aggregation. Platinum(II) 5,10,15,20-tetrakis(/>-carboxyphenyl)porphyrin and platinum(II)coproporphyrin-I ((36) for Pd read Pt) have been studied as phosphorescent labels of antibodies for use in time-resolved microscopy.189... 

Since endocytosis ofLDH was confirmed by TEM images (Figure 13.9), forthe next step, its specific endocytic pathway for membrane entry was determined by immunofluorescence and confocal microscopy. Cells were incubated with LDH-FITC, fixed with 3.7% freshly made formaldehyde, and then stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to the red fluorescent dye Texas Red (TR). The confocal microscopic images showed that green fluorescent... 

IHC stains as used for brightfield microscopy are sandwich procedures, consisting of sequential staining steps, each of which contributes amplification of the original signal. Specificity of the stain is provided by the primary antibody. The primary antibody also contributes to the sensitivity of the stain, but sensitivity is also the combination of each of the other steps of the stain protocol. It is critical that the steps used to amplify the signal in the stain process are not overdone, as these amplification steps can easily produce excess back-... 

Fig. 26 MR images of tumors of mice after they were injected with (a) paramagnetic av[33-specific RGD-liposomes and (b) nonspecific paramagnetic RAD-liposomes. (c, d) Fluorescence microscopy of 10 pm sections from dissected tumors revealed a distinct difference between tumors of mice that were injected with RGD-liposomes (c) or RAD-liposomes (d). Vessel staining was done with an endothelial cell-specific FITC-CD31 antibody. The red fluorescence represents the liposomes and the green fluorescence represents blood vessels. RGD-liposomes were exclusively found within the vessel lumen or associated with vessel endothelial cells (c), whereas RAD-liposomes (d) were also found outside blood vessels within the tumor (Adapted from [88])...

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

One particularly novel carrier was reported to consist of 50-70 nm colloidal gold particles of the type often used in cytochemical labeling techniques for microscopy (Pow and Crook, 1993) (Chapter 24). Adsorption of peptide antigens onto gold and subsequent injection of the complex into rabbits in an adjuvant mixture resulted in rapid production of antibody of extremely high titer. The resultant antibodies could be used in immunocytochemistry at dilutions from l-in-250,000 down to l-in-1,000,000, which is orders-of-magnitude beyond the dilutions typically used with lower-titer antibodies. 

Colloidal gold-labeled (strept)avidin can be used as highly sensitive detection reagents for microscopy techniques (Cubie and Norval, 1989) (Chapter 24). Finally, cytotoxic substances coupled to (strept)avidin can be used to direct cell-killing activity toward a tumor-cell-bound, biotinylated monoclonal antibody (or other targeting molecule) for cancer therapy (Hashimoto et al, 1984) (Chapter 21). 

An ordered antibody array has also been assembled on the solid surface by a combination of Langmuir Blodgett (LB) film method and self-assembling method. An ordered monolayer of protein A is deposited on the solid surface by LB method, which is followed by self-assembling of antibody. Individual antigen molecules which are complexed with the antibody array have been quantitated selectively by atomic force microscopy (AFM). 

Antibodies against sugars (carbohydrate residues) can be difficult to obtain and lectins are a solution to these problems. Lectins are naturally occurring plant and animal proteins or glycoproteins that selectively bind noncovalently to carbohydrate residues. Lectins can be labeled directly or secondary antibodies against lectins enables the use of other immuno techniques (30) including electron microscopy (31). 

Labeling antibodies with colloidal gold was developed for electron microscopy, but the procedure can be used for light microscopy as well. 

Knox RB, Clarke AE. Localization of proteins and glycoproteins by binding to labeled antibodies and lectins, in Electron Microscopy and Cytochemistry of Plant Cells (Hall JL, ed.), Elsevier-North Holland, Biomedical Press, Amsterdam, 1978, pp. 149-185. 

---