The labeling
Principle of indirect and direct labeling
Amplification of the fluorescence signal
Labeling by nick-translation or PCR
Counterstaining of chromosomes and nuclei
Fluorochromes
Antifade
Principle of indirect and direct labeling
As already introduced, Biotin-dUTP still serves as the most attractive non-radioactive label for FISH. Generally, for detection of the probe Avidin is used conjugated to a variety of different fluorochromes covering the entire visible spectrum of the light as well as some fluorochromes that emit in the near infrared. Since the probe is labeled with a hapten that itself is not fluorescent this procedure is called "indirect" detection (Figure 9). Other "indirect" labels include haptens like Dinitrophenol, Digoxigenin, or Oestradiol that can be detected by fluorochrome labeled antibodies. Again, antibodies against these haptens are now available that are coupled to numerous different fluorescent dyes.
"Direct" labels are dUTPs directly coupled to fluorochromes. After incorporation into the probe and following in situ hybridization the probe is visible without further need of detection procedures. Thus, a probe labeled for example with Cy3™ -dUTP (see below) is immediately visible after hybridization as a red fluorescence in the microscope (Figure 9). Again, a full range of "direct" labeled dUTPs are commercially available covering the entire visible spectrum of the light as well as the far red/close infrared. In many instances, the intensity of the fluorescence is not as strong as those provided by "indirect" systems. Since the workload in the "wet lab" to visualize the probe is much less compared to "indirect" labels and many FISH labs use electronic imaging of the signals, signal intensity may not be too important anymore. However, when analyzing very small probes "indirect" labels are still preferred.
Illustration of FISH with probes labeled indirectly with a hapten or directly with a fluorochrome. In FISH experiments with indirect labels (left side) such as Biotin or Digoxigenin the hapten itself is non-fluorescent. The probe is hybridized to the chromosomal DNA. After hybridization the signal becomes visible after application of a fluorescent antibody against the hapten or by fluorochrome labeled Avidin that binds to Biotin. FISH with direct labels uses fluorescent dUTPs incorporated in the probe (right side). With this technique there is no further need for detection of the hybridized probe after hybridization.
The fluorescence signal can be amplified by applying a "sandwich" technique of further fluorescent labeled antibodies. This can be used for direct as well as for indirect detection techniques. For example a fluorescent labeled antibody against Avidin can be used for signal amplification of the fluorescence emitted by the molecule bound to Avidine. Fluorescent labeled antibodies are also available that are directed against fluorochromes, thus amplifying the hybridization signal. Many of these systems, however, also amplify the "noise" of unspecific background fluorescence and in most instances a single "indirect" or "direct" label will give sufficient signal intensity.
Labeling by nick-translation or PCR
Hapten- or fluorescent labeled dUTPs are generally incorporated into the probe by either nick-translation or PCR. During nick-translation the double stranded probe is "nicked" by DNAse and partially digested. At the same time, the strand is filled up by a polymerase that incorporates dNTPs and also labeled dUTP offered in the labeling reaction. Nick-translation is usually used for labeling of cloned DNA such as BACs and YACs. However, PCR can also be used to lable these probes with degenerated primers (DOP-PCR). Usually DOP-PCR is used to generate and label complex probes such as paints from flow sorted chromosomes or micro-dissection. Most haptens and fluorochromes can withstand the high temperature cycles during PCR. PCR has the advantage that the entire probe is also being amplified. There are, however some haptens such as oestradiol-dUTP or some fluorochromes in the infrared that are not suitable for PCR labeling.
Counter staining chromosomes and nuclei
For FISH experiments, chromosomes and cell nuclei are generally counter stained by a fluorescent dye that is specific for DNA. The most common dye is DAPI (4´, 6-Diamidino-2-phenyl-indol) that is excited in the UV and chromosomes and cell nuclei show a bright blue fluorescence. Another common counter stain is propidium iodide that shows a red fluorescence. In experiments where several different probes should be visualized the best choice for a counter stain is DAPI.
Fluorochromes
The most frequent used dyes for the detection of the FISH signals are FITC (fluorescein isothiocyanate) that emits a green fluorescence and dyes with orange or red fluorescence, such as Cy3™ , or TexasRed™. Another commonly used fluorochrome in FISH experiments is Cy5™ that emits in the far red/close infrared. Since this fluorescence is not visible by eye it would need detection by an infrared sensitive camera mounted on the microscope. There are various other dyes with similar characteristics available from various different vendors. For example, FITC can well be replaced by Rhodamin 110, and Cy3™ by TAMRA (carboxytetramethyl- rhodamine) depending on the actual price and for some dyes, depending on the quality of the currently distributed lot.