Immunoelectron microscopy,

Affinity chromatography using factor XII as ligand leads to purification of u-PAR rather selectively, with only trace quantities of cytokeratin 1 or gClqR present [K. Joseph and A. Kaplan, unpubl. observations]. It is of interest that none of these three proteins possesses a transmembrane domain but u-PAR has a phos-phatidylinositol linkage within the cell membrane. Nevertheless, each of them has been isolated from purified cell membranes and they have been demonstrated to exist within the cell membrane by immunoelectron microscopy [41] presumably... 

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). 

Saito N, Konishi K, Takeda H, et al. Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions. J. Histochem. Cytochem. 2003 51 989-994. 

Tinglu, G., Ghosh, A., and Ghosh, B.K. (1984) Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold. J. Bacteriol. 159, 668. 

When the light microscope (LM) alone is not sufficient to study a system, the binding of an electron-dense ligand to an antibody facilitates the use of an EM. There are times when, in addition to EM study, immunoelectron microscopic analysis is necessary. Observed anatomical changes often lead to inquiries on the molecular events. This proved true in the case of the report by Inada et al. (22) in which they described a three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles. Immunoelectron microscopy was used to determine the behavior of cellular DNA during senescence. The procedure employed by the investigators is detailed below. 

Fig, 2. Representation ofthe central dogma ofmolecular biology. Immunoelectron microscopy accompanies many other techniques used to study gene expression including in situ hybridization. 

Molecular biology involves the study of the major macromolecules, DNA, RNA, and protein. The central dogma ofmolecular biology is illustrated in Fig. 2. The central dogma shows the relationship among the macromolecules in the processes of transcription and translation. Figure 2 also gives the relationship between immunoelectron microscopy and in situ hybridization. In situ hybridization allows one to localize a specific nucleic acid sequence. Immunoelectron microscopy is an essential component to the technique of in situ hybridization when applied at the EM level. 

Immunoelectron microscopy is not limited to nucleic acid localization but is also an essential component in the localization of a specific protein, polysaccharide, or theoretically any hapten under study. Therefore, immunoelectron microscopy is a valuable tool when it comes to the study of gene expression. Electron microscopy is a valuable tool in molecular biology and is even more powerful when combined with immunochemical techniques. 

Geuze H, Slot J, Van Der Ley P, Scheffer R. Use of colloidal gold particles in doublelabeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol 1981 89 653-665. 

Yamaguchi M, Kondo I. Immunoelectron microscopy of Proteus vulgaris by the plasma polymerization metal-extraction replica method differential staining of flagellar (H) and somatic (0) antigens by colloidal golds. J Electron Microsc 1989 38 382-388. 

Other recently developed techniques, such as immunoelectron microscopy (Chapter 18), electron systems imaging, and X-ray microanalysis (Chapter 19), should become routine practice in most EM laboratories and, thus, mainstays rather than ancillary EM methods. 

Stirling JW, Graff PS (1995) Antigen unmasking for immunoelectron microscopy labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium. J Histochem Cytochem 43 115 123... 

Roberg, K., 2001, Relocalization ofcathepsin D and cytochrome c early in apoptosis revealed by immunoelectron microscopy. Lab. Invest. 81 149-158 Roberg, K., Johansson, U., and OUinger, K., 1999, Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress. Free Radio. Biol. Med. 27 1228-1237 Roberg, K. and OUinger, K., 1998, Oxidative stress causes relocation of the lysosomal... 

Type XVI collagen is composed of 10 collagenous domains (COLI-COLIO) flanked by 11 NC domains. Type XVI collagen localizes near the dermal-epidermal junction. From immunoelectron microscopy of the papillary dermis, type XVI collagen associates with a fibrillin-1-containing matrix but not on collagen fibrils. ... 

Krenacs, T. and Krenacs, L. (1994) Immnnogold-silver staining (IGSS) for immunoelectron microscopy and in mnltiple detection affinity cytochemistry, in... 

Granzier H, Hehues M, Trombitas K. Nouuuiform elasticity of titiu in cardiac myoc3des a study using immunoelectron microscopy and cellular mechanics. Biophys J 1996 70 430-442. 

Positions of a few proteins located by immunoelectron microscopy and three positions in the 16S RNA are marked. The puromycin binding site labeled Pm was mistakenly thought to be near the peptidyltransferase center. (B) The 50S subunit. Only a... 

Many specific parts of ribosomal RNA molecules and specific proteins within the intact ribosome were located prior to the determination of high resolution crystal structures. One major approach was the use of immunoelectron microscopy. Antibodies to specific ribosomal proteins or to special sites in the RNA were prepared, and electron microscopy was used to map the binding sites of the antibodies on the ribosomal... 

The peptidyltransferase site. The position was located by binding of derivatives of the antibiotic puromycin (Fig. 29-13). An arylazide derivative of puromycin was photochemically linked (Eq. 23-27) to proteins L23, L18/22, and L15 immunoelectron microscopy, using antibodies to the -dimethyl-adenosine of puromycin,165 166 located the binding site adjacent to the central protuberance between the 50S subunit and 30S subunit near S14.5 4-Thio-dT-p-C-p-puromycin was photochemically crosslinked to G2553... 

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