Protocol Overview

1. Currently, we are working with four commonly used probe labels,
   • Digoxigenin (DIG)
   • Biotin (BIO)
   • Dinitrophenyl (DNP)
   • Fluorescein (FITC)
   and using standard immunofluorescence techniques to detect these labels. For each label, there are multiple ways to achieve the fluorescent visualization, but for the sake of simplicity, the diagram shows only one way for each label.
2. Sequential tyramide signal amplification (TSA) reactions using horseradish peroxidase (HRP) conjugates give very sensitive and reliable double fluorescent staining.
3. Fluorescent secondary antibodies (2nd Ab.) can be used to label primary antibodies (anti-Hap Ab.) against the probe labels, or 'haptens'.
4. 'Zenon' (Molecular Probes) refers to a method of making fluorescently labeled antibody complexes, which are then incubated with embryos. Not only does this method eliminate the need to maintain species separation with primary antibodies, but also makes it possible to visualize hybridized probes with only one incubation and washing step.
5. We have also used directly labeled (DL) probes, into which fluorescent dye molecules of the 'Alexa' series (Molecular Probes) have been incorporated. These probes can be visualized on embryos immediately after hybridization and washing, but the signals produced are considerably less than with the other methods.
6. For more details on sequential TSA, Zenon antibody labeling, and direct label probes, see Alternative Methods for Fluorescent Detection.
7. It is possible to combine any and all of these methods to get a multi-color fluorescent stain, providing great flexibility in the design of experiments. A more detailed discussion of them can be found here under Conclusions and Future Problems.

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