DIG-probes

To the second universal add 22 pL of NBT + 17 pL of X-phosphate 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) for both biotin and dig probes. Wrap both universal tubes in tinfoil to keep out the light. 

Phenol extraction of biotinylated or DIG-probes should always be avoided due to the possible partitioning of the probe into the phenol layer. Similarly, NACS resins should not be used for the purifica-... 

LtL/30-mm cover slip 2-6 oL dig-probe/50 pL 1 milhon cpm/30 mm cover slip O.Sxtotal volume... 

J.L. Wise, P.K. Kammer, and J.A Murray, Post-test calibration of single-use, antimony, 24-hour ambulatory esophageal pH probes is necessary. Dig. Dis. Sci. 49, 688-692 (2004). 

For double labeling, McFadden suggests labeling one probe with dig-oxygenin and the other with biotin ... 

ZytoVision ZytoDot2C SPEC probes are labeled with DIG and DNP haptens and the probe hybridization sites can be visualized with a BISH detection kit including anti-DIG and anti-DNP 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... 

ZytoVision ZytoDot2C GISH Implementation Kit contains all necessary BISH detection reagents for the detection of DIG-and DNP-labeled DNA probes on formalin-fixed tissue sections. The probes can be purchased from ZytoVision or other vendors. 

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. 

Prepare Hybridization Mix containing 500 ng/ml of DIG-labeled RNA probe in a plastic screw-cap tube, and transfer the embryos. 

Fig. 1. Dual nucleic acid detection. The target DNAs (A and B) are denatured and hybridized with complementary DNA probes labeled with biotin (Bio ), or digoxigenm (Dig ). Biotin and digoxigenm residues are detected, respectively, with avidin peroxidase (red) and antibody to digoxigenin labeled with alkaline phosphatase (blue).

Figure 4.5 Digoxigenin (DIG) labeling of hybridization probes (from Roche hybridization manual).

One of the most sensitive method for detecting probe-target hybrids involves an alkaline phosphatase-conjugated anti-DIG antibody and chemoluminescent alkaline phosphatase substrates (Roche, Molecular Diagnostics). Following hybridiza-... 

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