6 In Situ Hybridization for DNA: Fluorescent Probe
69
2. Dehydrate specimen slides in ethanol series in order to maintain denatured DNA single strands in specimen.
3. Make hybridization mix and denature the hybridization mix
at 74°e for 5 mins.
4. Apply the denatured hybridization mix to specimen target
area on slides.
5. Hybridize probe DNA to target specimen DNA at 37°e for 30
mins to 2 days.
6. Wash to remove non-specifically bound probe in 50% formamide/2X sse at 46°e, followed by buffer wash. (If the probes
are modified with haptens, such as biotin or digoxigenin etc.,
this step should be followed by fluorescent staining procedures.)
7. Air dry slides, and apply counterstain.
8. View using fluorescence microscope with a suitable filter set.
Slides containing cells and chromosomes are prepared using
standard technique with methanol-acetic acid (3:1) fixation.
Paraformaldehyde (1-4% in phosphate buffer, pH 7.4, for 1030 mins at 4°C) or ethanol fixation also yields good signals. However, the signals are much better when the cells do not have cytoplasm. After fixation, slides can be stored at -20 o e for up to 6
months.
Formalin-fixed paraffin embedded tissue sections are also
used for FISH assay. After deparaffinization and rehydration,
the sections are treated as described in standard FISH protocol
using pepsin or other proteolytic enzymes at high concentration
before or after denaturation. The digestive conditions vary
among specimen types, and need to be optimized in each case.
Materials
- Fluorescent microscope (e.g., Olympus BX type, Nikon) with
suitable single-bandpass or multi-bandpass filter sets (for
FITe, Rhodamin, Texas-Red, Spectrum-Orange, SpectrumGreen, DAPI, PI, etc.).
- Water Baths: 37°e, 46°e and 74°e
- Vortex
69
2. Dehydrate specimen slides in ethanol series in order to maintain denatured DNA single strands in specimen.
3. Make hybridization mix and denature the hybridization mix
at 74°e for 5 mins.
4. Apply the denatured hybridization mix to specimen target
area on slides.
5. Hybridize probe DNA to target specimen DNA at 37°e for 30
mins to 2 days.
6. Wash to remove non-specifically bound probe in 50% formamide/2X sse at 46°e, followed by buffer wash. (If the probes
are modified with haptens, such as biotin or digoxigenin etc.,
this step should be followed by fluorescent staining procedures.)
7. Air dry slides, and apply counterstain.
8. View using fluorescence microscope with a suitable filter set.
Slides containing cells and chromosomes are prepared using
standard technique with methanol-acetic acid (3:1) fixation.
Paraformaldehyde (1-4% in phosphate buffer, pH 7.4, for 1030 mins at 4°C) or ethanol fixation also yields good signals. However, the signals are much better when the cells do not have cytoplasm. After fixation, slides can be stored at -20 o e for up to 6
months.
Formalin-fixed paraffin embedded tissue sections are also
used for FISH assay. After deparaffinization and rehydration,
the sections are treated as described in standard FISH protocol
using pepsin or other proteolytic enzymes at high concentration
before or after denaturation. The digestive conditions vary
among specimen types, and need to be optimized in each case.
Materials
- Fluorescent microscope (e.g., Olympus BX type, Nikon) with
suitable single-bandpass or multi-bandpass filter sets (for
FITe, Rhodamin, Texas-Red, Spectrum-Orange, SpectrumGreen, DAPI, PI, etc.).
- Water Baths: 37°e, 46°e and 74°e
- Vortex