Immunofluorescence microscopy,

The principal molecular constituent of thin filaments is actin. Actin has been highly conserved during the course of evolution and is present in all eukaryotes, including single-celled organisms such as yeasts. Actin was first extracted and purified from skeletal muscle, where it forms the thin filaments of sarcomeres. It also is the main contractile protein of smooth muscle. Refined techniques for the detection of small amounts of actin (e.g., immunofluorescence microscopy, gel electrophoresis, and EM cytochemistry) subsequently confirmed the presence of actin in a great variety of nonmuscle cells. Muscle and nonmuscle actins are encoded by different genes and are isoforms. 

Proteins that cross-link actin filaments bind to their sides to produce bundles or three-dimensional networks (Otto, 1994). In microvilli, approximately 20 actin filaments of the core are cross-linked by villin (95 kD) and fimbrin (68 kD) in helical array to form a compact bundle (Figure 5). Filamin (2 x 250 kD) induces the formation of an actin network with gel formation. By immunofluorescence microscopy, this ABP is found in the ruffled, motile edge of cultured cells, where only actin filaments are abundant. 

In the last decade, modem biochemical methods have been used for analysis of protein binders [20,21] in one case a group led by A. Heginbotham identified egg proteins in a seventeenth century painting using immunofluorescent microscopy and enzyme-linked immunosorbent assay (ELISA) [20]. 

A. Heginbotham, V. Millay, M. Quick, The use of immunofluorescence microscopy (IFM) and enzyme linked immunosorbent assay (ELISA) as complementary techniques for protein identi fication in artists materials, J. Am. Inst. Cons., 45, 89 105 (2006). 

Figure 11.2 Morphological differences between human alveolar epithelial cells in primary culture (A and C) and the A549 cell line (B and D). Cells are visualised by light microscopy (A and B) and immunofluorescence microscopy (C and D) using an antibody against a tight junctional protein, occludin.

Melan, M. A. and Slnder, G. (1992) Redistribution and differential extraction of solnble proteins in permeabilized cnltnred cells implications for immunofluorescence microscopy. J. Cell Sci. 101, 731-743. 

Weber, K., Rathke, P. C., and Osborn, M. (1978) Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells viewed by electron microscopy and by immunofluorescence microscopy. Proc. Natl. Acad. Sci. USA 75, 1820-1824. 

Wick, S. M., Seagull, R. W., Osborn, M., Weber, K., and Gunning, B. E. S. (1981) Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J. Cell Biol. 89, 685-690. 

The pioneering immunofluorescence studies of Albert Coons and colleagues in the 1940s and 1950s (reviewed in ref. 11) established the effectiveness of fluorescein for immunofluorescence microscopy. The green emission of fluorescein isothiocyanate (FITC) was shown to provide a strong signal, well separated from blue cellular autofluorescence. Continued wide use of fluorescein attests to its utility. 

Small, J. V., Zobeley, S., Rinnerthaler, G., and Faulstich, H. (1988) Coumarin-phalloidin a new actin probe permitting triple immunofluorescence microscopy of the cytoskeleton. J. Cell Sci. 89,21-24. 

The association between a histone tail modification and a particular functional state of chromatin, came with the demonstration that transcriptionally active chromatin fractions were enriched in acetylated histones, firstly by biochemical co-fractionationation ([8,9] and references therein) and then by Chromatin ImmunoPrecipitation, ChIP [10]. Subsequently, regions of transcriptionally silent constitutive and facultative heterochromatin, were shown, by immunofluorescence microscopy, to be under-acetylated [11,12]. This supported the idea that acetylation of the histone tails, with the associated loss of positive charge and reduction in DNA-binding constant, somehow caused chromatin to become more open (or less condensed ) and thereby more conducive to transcription. While this is likely to be an important contributory factor, it has now become clear that the... 

Further improvement of microchemical methods for proteinaceous media was based on immunological techniques. The high specificity of the antigen-antibody reaction enables the discrimination of the same protein coming from different species, or the detection of multiple antigens in the same sample. Application to the analysis of artwork has been reported in two types of immunological techniques immunofluorescence microscopy (IFM), and enzyme-linked immunosorbent assays (ELISA) [31]. 

Wolbers R, Landrey G (1987) The use of direct reactive fluorescent dyes for the characterization of binding media in cross sectional examinations. Preprints of 15th Annual Meeting of the American Institute for Conservation and Artistic Works, Vancouver, 168-202. Heginbotham A, Millay V, Quick M (2006) The use of immunofluorescence microscopy and enzyme-linked immunosorbent assay as complementary techniques for protein identification in artist s materials. J Am Inst Conserv 45 89-105. 

Another approach employed to increase sensitivity of microarrays is signal amplification at the post-hybridization stage. One such technique is fyramide signal amplification (TSA) which requires 20-100 times less RNA than direct cDNA labeling (40). This method was originally used in immunohistochemistry and has been an important tool for immunofluorescence microscopy (41,42). 

No evidence has been found for the mitosomal genome in any organism so far. An earlier study on E. histolytica reported the presence of DNA in Entamoeba mitosomes (Ghosh et al. 2000) and synonymized these organelles with DNA-containing structures named kinetoplast-like organelles (EhKO) (Orozco et al. 1997). However, work by Leon-Avila and Tovar (2004) later showed that EhKO and mitosomes are not related structures. Moreover, in situ nick translation coupled with immunofluorescence microscopy failed to detect the presence of DNA in Entamoeba mitosomes (Leon-Avila and Tovar... 

Fig. 4 Immunofluorescence microscopy of Giardia intestinalis showing segregation of mi-tosomes during mitosis. A Interphase, prophase, and C telophase cells. Nuclei were stained by DAPI (blue), mitosomes were detected by an antibody raised against GiiscU (red), and axonemes were visualized by the antibody AXO 49 recognizing polyglycylated carboxy-terminal peptides of a- and fl-tubulin (green). Note the proximity of mitosomes (white arrows) to axonemes...

Pollenz RS, Sattler CA, Poland A. 1994. The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa lclc7 cells by immunofluorescence microscopy. Mol Pharmacol 45 428-438. 

In immunofluorescence microscopy, fluorescent compounds (which absorb light at the exciting wavelength and then emit it at the emission wavelength) are attached to an antibody specific for the subcellular structure under investigation. The antibody is then added to the specimen and allowed to bind. Unbound antibody is removed and the specimen is illuminated at the exciting wavelength, to visualize where the antibody has bound. 

This variation of immunofluorescence microscopy uses a laser to focus light of the exciting wavelength on to the specimen so that only a thin section of it is illuminated. The laser beam is moved through the sample, producing a series of images which are then reassembled by a computer to produce a three-dimensional picture of the specimen. 

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