Antibodies fluorescence microscopy

Cells separated by filtration from large aliquots of water can be visualized and counted on a 0.25 xm filter using the epifluorescent technique and a stain such as acridine orange. Stains such as fluorescein diacetate, 5-cyano-2,3-ditolyltetrazolium chloride or p-iodonitrotetrazolium violet indicate active metabolism by the formation of fluorescent products [9]. Antibody fluorescence microscopy is similar to general fluorescent microscopy, except that the fluorescent dye used is bound to antibodies specific to SRBs. Only bacteria recognized by the antibodies fluoresce. Results can be analyzed within two hours. The technique detects viable and nonviable bacteria but it is limited to the type of SRB used in the manufacture of these antibodies [19]. 

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... 

Fluorophores were introduced to fluorescence microscopy in the early twentieth century, but did not see widespread use until the early 1940s when Albert Coons developed a technique for labeling antibodies with fluorescent dyes, thus giving birth to the field of immunofluorescence (http //www.olympusconfocal.com/ theory/fluorophoresintro.html). By attaching different fluorophores to different antibodies, the distribution of two or more antigens can be determined simultaneously in the tissue section and, in contrast to brightfield microscopy, even in the same cells and in the same cell structures (see Chap. 8). 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

Fluorophore absorbs ultraviolet light (or violet, blue or green) and emits light of longer wavelength. Fluorophores are used in immunohistochemistry for labeling primary or secondary antibodies in direct and indirect immunostain-ing methods, respectively. They can be visualized in fluorescence microscopy using special filter sets. 

C. L. Poglitsch and N. L. Thompson, Interaction of antibodies with Fc receptors in substrate-supported planar membranes measured by total internal reflection fluorescence microscopy, Biochemistry 29, 248-254 (1990). 

C. L. Poglitsch and N. L. Thompson, Substrate-supported planar membranes containing murine antibody Fc receptors A total internal reflection fluorescence microscopy study, in Biosensor Technology, Fundamentals and Applications (R. P. Buck, W. E. Hatfield, M. Umafiia, and E. F. Bowden, eds.), pp. 375-382, Marcel Dekker, New York (1990). 

The immunocytochemical staining of cytochrome C offers another alternative since, upon exposure to apoptotic stimuli, cytochrome C is rapidly released into the cytosol, an event that may be required for the completion of apoptosis in some systems (L2). The effect of cytosolic cytochrome C is thought to be the activation of caspases. The immunocytochemical staining of cytochrome C localized in mitochondria in healthy cells or diffused in the cell cytoplasm with monoclonal antibody (Promega) after induction of apoptosis, as detected by fluorescence microscopy, can be used for monitoring apoptosis (L2, M7, S8, T6). It is also a simple, rapid, specific mefhod for quanfifafive assessment of apoptotic cells. 

Immunoassay kit, where the antibodies of 8-oxoguanine (96) are conjugated with fluorescein isothiocyante (97) as fluorophore and combine with the oxidized DNA. Detection of the greenish fluorescence is by fluorescence microscopy for tissues or by fluorescence-activated ceU sorting for cell suspensions . 

Successful cell surface display of the protein can be verified by inducing gene expression via addition of anhydrotetracycline to the culture medium and immunofluorescence staining of the cells using an antibody directed against the protein to be displayed (Protocol 1). Analysis can be performed by fluorescence microscopy or flow cytometry. 

Proteomic Fluorescence microscopy In situ visualisation of cellular/ECM protein with fluorescence-labelled antibody Multiplexing capability (typically 3 fluorophores). Extra- and intracellular antigen location on opaque biomaterials. Issues with bleaching and autofluorescence. Yes/no... 

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