FITC-labeled probe

Dako DuoCISH Kit contains HRP- and AP-based immunological detection reagents for Texas Red- and FITC-labeled probes. This is an ideal kit for manual dual-color BISH assay detection. 

Probes can be differently labeled with hapten labels, for example carboxyfluorescein (6-FAM), digoxigenin (DIG) and biotin can be bound to LNA oligos. The choice of probe label depends on experimental design and the techniques available in the laboratory. The hapten label provides a template for crucial signal amplification since the FITC label on the oligo itself is not sufficient to allow detection in standard epifluorescence. In this study, the fluorescence signal was obtained with the TSA-FITC substrate, which allowed detection of miR-21 and miR-205. 

Hybridisation of FITC-labelled target DNA to biotinylated probes on streptavidin-modified SPCEs. Addition of anti-FITC-ALP conjugate Hybridisation of Pt(II) complexed target DNA to biotinylated probes on streptavidin-modified SPCEs... 

In another report, detection of hybridization of an unlabeled target was achieved by exploiting laminar flows in microchannels. Two streams of FITC-labeled oligonucleotide probe and its unlabeled complementary target were... 

Figure 4. Staining with CEN-18 FITC-labeled PNA probe (green signals). Arrows illustrate the localization preference close to the membrane of gene-poor DNA domains.

Fig. 4. Carbohydrate microarrays containing 22 carbohydrate probes after incubation with (a) FITC-labeled Con A, (b) FITC-labeled N. pseudonarcissus and (c) FTTC-labeled wheat germ agglutinin.

PCR-ELISA is a hybrid assay combining PCR as a first step and using ELISA as a detection system for the amplicons (Figure 9.1). The PCR step produces biotinylated amplicons that are bound further to a streptavidin-coated microtiter plate. The amplicons are denatured to produce single-stranded PCR products, and only the biotinylated strand stays in the well of the microtiter plate. A FITC-Iabeled probe is bound to the PCR strand, a step that adds an extra level of specificity to the assay. An enzyme-labeled anti-FITC antibody is used to produce a colorimetric signal that can be measured by an ELISA reader. This method has been applied successfully to the detection of <10 ppm hazelnut in processed foods and food ingredients, which eliminated the potential for cross-reactivity observed in ELISA (Holzhauser et al., 2002). 

Direct method in which the probe-target hybrid can be visualized after the hybridization. When fluorochrome-labeled probes are used, the visualization can be achieved by fluorescence microscopy. Various fluorochromes with different emission colors are now available such as AMA, FITC, fluorescein, rhodamine CY3 and Texas red. The use of different probes labeled by different fluorochromes... 

In a report by D.G. Shchukin, the release and reloading cycles of a PPy microcontainer system were visualized by fluorescence microscopy, employing FITC-labeled dextran as a probe (Figure 11.14). The initial dextran-loaded PPy microcontainer electrode demonstrated a high uptake yield in solution without applied potential bias. Changing to a reductive potential resulted in the release of the encapsulated material. Further, hollow polyelectrolyte capsules could be reloaded with new portions of FITC-dextran at a positive potential bias. Twenty minutes of incubation at an oxidative potential were sufficient to reload the capsules inside the PPy microcontainer electrode. The incubation time or release time could be reduced by increasing the potential bias. 

Polyelectrolyte capsules reinforced by inorganic nanoparticles are able to incorporate internally, in controlled manner, a variety of different organic as well as bioor-ganic materials (drugs, polymers, enzymes, etc.) [58], In this respect, YF3/PAH capsule permeability and release properties were studied by employing fluorescein isothiocyanate (FITC)-labeled dextran of 2000 kDa molecular weight as a probe. 

Fluorescein-5-isothiocyanate (FITC) labelling (fluorescence emmission 485 nm, extinction 535 nm) was done according to Maeda et aP. and Faraday et aPl Texas Red and Texas Red labelled albumin are commercially available from Molecular Probes, Leiden, Netherlands. Dansylchlorid labelling of proteins was done according to Miekka and Ingham ... 

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

In addition to the wide range of commercial probes, many other fluorescent molecules have been synthesized and described in the literature. Only a handful, however, are generally used to label antibody molecules. Perhaps the most common fluorescent tags with application to immunoglobulin assays are reflected in the main derivatives produced by the prominent antibody manufacturing companies. These include derivatives of cyanine dyes, fluorescein, rhod-amine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 20.16 shows the reaction of fluorescein isothiocyanate (FITC), one of the most common fluorescent probes, with an antibody molecule. 

The following protocol is a generalized method for labeling amine-modified oligonucleotides with a fluorescent probe, such as FITC. It is based on the method of Morrison (1992). 

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. 

Abnova FISH probes and Dako FISH probes are labeled with fluorescein isothiocyanate (FITC) and Texas Red (sulforhod-amine lOI acid chloride) haptens, and the probe hybridization sites can be visualized with a BISH detection kit including anti-FITC and anti-Texas Red antibodies. 

Common haptens used for labeling DNA probes for BISH assays are biotin, DIG, DNP, FITC, and Texas Red. Based on the size of your DNA targets, you may choose from a direct detection or an indirect detection for BISH assays. In general, an indirect detection system can provide better sensitivity compared to a direct detection system. For an indirect detection, you need to select a combination of two antibodies raised with two different animal species, such as a mouse anti-DIG antibody and a rabbit anti-DNP antibody, so that enzyme-labeled anti-mouse antibody and anti-rabbit antibody can be applied for signal detection. If a direct BISH detection is going to be applied, anti-hapten antibodies raised in the same animal species that are labeled with either AP or HRP enzyme molecules... 

The second label also may be a fluorescent compound, but does not necessarily have to be. As long as the second label can interact with the emission of the first label and modulate its signal, binding events can be observed. Thus, the two labeled DNA probes interact with each other to produce luminescence modulation only after both have bound target DNA and are in enough proximity to initiate energy transfer. Common labels utilized in such assay techniques include the chemiluminescent probe N-(4-aminobutyl)-N-ethylisoluminol and the fluorescent compounds FITC (Chapter 8,Section 1.1), TRITC, and Texas Red sulfonyl chloride (Chapter 8, Section 1.2). Fora review of these techniques, see Morrison (1992). 

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