Immuno-fluorescence

Huang, S. N., Minassian, H., and Moore, J. D. (1976) Application of immuno-fluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest. 35, 383-390. 

VieUdnd, U. and S wierenga, S. H. (1989) A simple fixation procedure for immuno-fluorescent detection of different cytoskeletal components within the same cell. Histochem. 91, 81-88. 

In this method, the primary antibodies are combined into a single cocktail and applied simultaneously in a single-reaction step. The same is done for the labeled secondary antibodies. Antibodies of different animal species or immunoglobulin isotypes (noncrossreactive) or autologous antibodies that are directly coupled to different enzymes/fluorochromes are required. Immuno-fluorescent methods are particularly suited for this method. 

Ward, B. B. (1982). Oceanic distribution of ammonium-oxidizing bacteria determined by immuno-fluorescent assay. Journal of Marine Research 40, 1155—1172. 

Bullous pemphigoid has been reported in an 84-year-old man after topical therapy with fluorouracil 1% solution daily over several days for actinic keratosis. All treated lesions became bullous, with the development of a few bullae on untreated areas of normal skin. Bullous lesions were pruritic and sore and some contained hemorrhagic fluid. There was a leukocytosis (11.7 x 10 /1). The blister fluid contained predominantly eosinophils, and immuno-fluorescent studies of the serum and blister fluid showed anti-basement membrane antibody titers of 1 640 and 1 160 respectively. Fluorouracil was discontinued and the patient was treated with steroids and saline compresses, with abatement of symptoms (119). 

A.G.J. Buma, E.J. van Hannen, M.J.W. Veldhuis, L. Roza, W.W.C. Gieskes (1995). Monitoring UV-B induced DNA damage in individual diatom cells by immuno-fluorescent thymine dimer detection. J. Phycoi, 31, 314-321. 

Poirier, M. C. Stanley, J. R. Beckwith, J. B. Weinstein, I. B. Yuspa, S. H. Indirect immuno fluorescent localization of benzo-a-pyrene adducted to nucleic-acids in cultured mouse keratinocyte nuclei. Carcinogenesis (Lond), 3 345-8. 1982. 

A two- to three-fold increase in Na -K -ATPase activity, a nonspecific marker a three-fold reduction in a negative marker, alkaline phosphatase identified with proximal tubular cells and immuno-fluorescent localization of Tamm-Horsfall protein, a specific marker for mTALH cells. 

The SLO is thawed in a 37° bath and 1 vol. 2 x buffer A is added. This solution is incubated 5 min at 37° to activate the SLO. After this incubation, the SLO is diluted to 2 U/ml with 1 x buffer A and kept in ice before being used within 30 min. The buffer A, the buffer B, and a 24-well plate with 70-90%-confluent cells are put on ice. The cells are washed twice with 500 A ice-cold 1 x buffer A, and 250 A 2 U/ml SLO are added. After a 10-min incubation in ice, the cells are gently washed with 500 /ul ice-cold 1 x buffer A. Following the addition of 500 //I ice-cold buffer B, the cells are transferred to a 37° bath. After a 5-min incubation, the cells are fixed and processed for immuno-fluorescence (Bonazzi et al., 2005). 

Figure 4. Indirect immuno-fluorescent technique for study of localization of components of the kallikrein-kinin system.

Lin, S., Feinstein, T.N., Zhang, H., and Carpenter, E.J. (2003) Development of an immuno-fluorescence technique for detecting Pfiesteria piscicida. Harmjul Algae, 2, 223-231. 

F.C. Gong, Z.J. Zhou, G.L. Shen, and R.Q. Yu, Schistosoma japonicum antibody assay by immuno-sensing with fluorescence detection using 3,3, 5,5 -tetramethylbenzidine as substrate. Talanta 58, 611-618 (2002). 

Immunoaffinity chromatography (IAC), 6 400—402 12 137, 145 Immunoanalyzers, automated, 14 150 Immunoassay(s), 14 135-159. See also Immunoassay- DNA probe hybrid assays Immunoassay methods Immuno(bio)sensors antibody-antigen reaction, 14 136-138 basic technology in, 14 138-140 chemiluminescent, 14 150-151 classification of, 14 140-153 design of, 14 139-140 enzyme, 14 143-148 fluorescence, 14 148-150 highly specific, 14 153 historical perspective on, 14 136 microarrays and, 14 156—157 microfluidics in, 26 968—969 monoclonal versus polyclonal antibodies in, 14 152-153... 

Fig. 11.2 Localization of GFP and calsequestrin (CSQ) in neonatal rat cardiac myocytes. Myo cytes were infected with a recombinant adenovirus containing the cDNAs of GFP and CSQ. Both cDNAs were expressed under control of a separate cytomegalovirus promoter. Expression of CSQ and the coexpressed GFP was detected by fluorescence microscopy. Left-. GFP fluorescence (green), middle immunostaining of CSQ (red), right overlay of GFP fluorescence and immunos taining. Nuclei were counterstained with DAPI (blue). Courtesy of Ulrich Gergs...

H2A-Bbd is found only in the active areas of the nucleus and displays an exclusive deposition pattern with mH2A [61]. Furthermore, fluorescence immuno-chemistry has shown that this H2A variant co-localized with acetylated H4 [59]. Although the structural properties of H2A-Bbd containing nucleosomes remain to be characterized, preliminary observations suggest that the particle may be specialized for activating transcription. 

Watts AG, Swanson LW 1987 Efferent projections of the suprachiasmatic nucleus II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immuno-histochemistry in the rat. J Comp Neurol 258 230—252 Xian CJ, Zhou X-F 1999 Roles of transforming growth factor-alpha and related molecules in the nervous system. Mol Neurobiol 20 157—183... 

Use of a surrogate end point that is quick and easy to obtain Permeation experiments using a radiolabeled, fluorescent, HPLC-detectable, or radio immuno assay/enzyme linked immuno sorbent assay-detectable marker necessitate the need of extensive sample handling and sample analysis. This accentuates the cost of sample analysis and overall time spent in characterizing the efficacy of formulations. Furthermore, current state of the art fluidics systems put a fundamental limit on the number of samples handled in a given time. 

A multiresidue preparation technique—MSPD—has also been applied to the analysis of CAP residues in meat samples. Two fractions were collected by elution with methylene chloride and ethyl acetate. No additional purification was necessary. Diode assay detection and fluorescence detectors were recommended for the multiresidue analysis of sulfonamides, benzimidazoles, nicarbazin, furazolidone, and CAP. The percentage recoveries and linearity of the method were evaluated. The method was linear from 50 to 250 /tg/kg of CAP. Not only do the authors recommend the MSPD multiresidue procedure for HPLC analysis, but it could be associated with several detection modes, such as immuno- or receptor assays. The MSPD technique represents a new approach in the field of biological-matrix extraction and provides a great possibility for the analysis of a wide range of compounds (20). 

Like in RILAs, an advantage of fluorescence detection is the possibility of developing homogeneous FILAs using direct or indirect (competitive or displacement) approaches. The fluorescence polarization immunoassay (PFIA) and their homolog fluorescence polarization immuno-like assay (PFILA) are two of the most widely used procedures in homogeneous fluoroassays. Both are based on the principle that fluorescence polarization gives a direct measure of the bound/free ratio of the labeled analyte (tracer) without the need for their separation [23, 28]. 

The avidin-biotin system was developed for detecting antigens at the electron microscope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposed the use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) proposed a variety of labeled avidin-biotin methods which were further supported by Warnke and Levy (1980). The avidin-biotin methods used today are similar to the system described by Hsu et al. (1981). This system is a significant improvement over the previous immuno-histochemical techniques. The problem of endogenous biotin is discussed on page 98. 

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