1 FISH Techniques, FISH Probes and Their Applications in Medicine and Biology - An Overview
17
After hybridization, unbound probe must be removed. This is carried out
by washing the slide in a saline solution (SSC - standard saline citrate)
where probe-target hybrids are stable. The specificity of this wash can
be regulated by altering one of the following parameters: (1) salt concentrations of washing solutions, (2) formamide concentrations, (3) temperature.
The following are common examples of hybridization and post-hybridization washing: (1) hybridization in 60% formamide, 2xSSC at 37°C
followed by washing in 60% formamide, 2xSSC at 42°C; or (2) hybridization in 50% formamide, 2x SSC at 37°C followed by washing in 0.1 x SSC at
37°C. It should be noted that formamide is a toxic organic solvent and
steps involving this compound must be performed under a hood.
After the fmal washing step the presence and localization of probe-target
hybrids can be demonstrated by detection of the labeled probe. Probes
labeled directly with fluorochromes can be visualized immediately by
fluorescence microscopy using appropriate filter sets for excitation and
emission. Probes labeled indirectly with biotin or digoxigenine can be
visualized by methods based on affinity (e.g. streptavidin coupled to a
fluorochrome) or immunodetection (fluorochrome-conjugated antibody
against digoxigenine), respectively. The streptavidin and antibody can be
coupled to either a fluorochrome or to an enzyme-substrate system.
Avidin can be used instead of streptavidin but this increases the risk
of unspecific background due to the affinity of avidin for cellular components. Fluorescence based approaches are particularly useful when high
resolution is required or when more than one sequence is hybridized
to the same tissue or cell preparation. In interphase cytogenetics, fluorescence based methods are particular useful as multicolor hybridization
techniques can be used (see below).
Before the samples are analyzed, the chromosomes or nuclei are most
often counterstained using either DAPI (4',6-diamidino-2-phenylindole)which emits blue light or propidium iodine which emits red light. DAPI
however, is the most commonly used counterstain because probes in general are labeled with FITC (green), rhodamin/Texas Red (red) or both if
two probes are hybridized simultaneously.
One important problem when using fluorescent based methods, is
bleaching or fading of the signal while being analyzed or left in the light.
This problem can be reduced by mounting the sample in so called antifade
solutions (Longin et al. 1993).
Non-fluorescent methods are used where high sensitivity is important.
Two enzyme-based systems are frequently used, namely the alkaline phosAd 5: Post-hybridization washing
Ad 6: Detection
17
After hybridization, unbound probe must be removed. This is carried out
by washing the slide in a saline solution (SSC - standard saline citrate)
where probe-target hybrids are stable. The specificity of this wash can
be regulated by altering one of the following parameters: (1) salt concentrations of washing solutions, (2) formamide concentrations, (3) temperature.
The following are common examples of hybridization and post-hybridization washing: (1) hybridization in 60% formamide, 2xSSC at 37°C
followed by washing in 60% formamide, 2xSSC at 42°C; or (2) hybridization in 50% formamide, 2x SSC at 37°C followed by washing in 0.1 x SSC at
37°C. It should be noted that formamide is a toxic organic solvent and
steps involving this compound must be performed under a hood.
After the fmal washing step the presence and localization of probe-target
hybrids can be demonstrated by detection of the labeled probe. Probes
labeled directly with fluorochromes can be visualized immediately by
fluorescence microscopy using appropriate filter sets for excitation and
emission. Probes labeled indirectly with biotin or digoxigenine can be
visualized by methods based on affinity (e.g. streptavidin coupled to a
fluorochrome) or immunodetection (fluorochrome-conjugated antibody
against digoxigenine), respectively. The streptavidin and antibody can be
coupled to either a fluorochrome or to an enzyme-substrate system.
Avidin can be used instead of streptavidin but this increases the risk
of unspecific background due to the affinity of avidin for cellular components. Fluorescence based approaches are particularly useful when high
resolution is required or when more than one sequence is hybridized
to the same tissue or cell preparation. In interphase cytogenetics, fluorescence based methods are particular useful as multicolor hybridization
techniques can be used (see below).
Before the samples are analyzed, the chromosomes or nuclei are most
often counterstained using either DAPI (4',6-diamidino-2-phenylindole)which emits blue light or propidium iodine which emits red light. DAPI
however, is the most commonly used counterstain because probes in general are labeled with FITC (green), rhodamin/Texas Red (red) or both if
two probes are hybridized simultaneously.
One important problem when using fluorescent based methods, is
bleaching or fading of the signal while being analyzed or left in the light.
This problem can be reduced by mounting the sample in so called antifade
solutions (Longin et al. 1993).
Non-fluorescent methods are used where high sensitivity is important.
Two enzyme-based systems are frequently used, namely the alkaline phosAd 5: Post-hybridization washing
Ad 6: Detection