Leica Kreatech FISH probes Mode D'emploi page 9

Kreatech FISH probes
Recommendations for fluorescence microscopy:
For optimal visualization use a well maintained and regularly
calibrated microscope equipped with a 100W mercury lamp or other
appropriate light source and a 63x or 100x fluorescence objective.
Triple band-pass filters (DAPI/FITC/Cy3 or DAPI/FITC/TRITC) are used
to view multiple colors, single band-pass filters are used for individual
color visualization.
Suitable excitation and emission range for Kreatech fluorophores:
Fluorophore
PlatinumBright™ 415
PlatinumBright™ 495
PlatinumBright™ 550
Troubleshooting
Check protein digestion and pre-treatment by applying 15 μl DAPI
counterstain and evaluate slides using a fluorescence microscope
equipped with a DAPI filter. A 15 min protein digestion is normally
sufficient for a wide range of breast tumors. Remove coverslip and
soak tissue in 2 x SSC, pH 7.4 for 2 min at RT. Prolong protein digestion
by 2 - 20 min if sample is not sufficiently digested. Use a fresh sample
and reduce protein digestion time if the sample is over-digested.
Alternative protocol for probe denaturation (separate probe and
sample denaturation):
1. Denature slide in 70% formamide / 2 x SSC, pH 7.0 at 72 °C (±1 °C)
for 2 min.
2. Dehydrate in ice cold (-20 °C) 70%, 85%, and 100% ethanol for
2 min each. Air-dry.
3. Denature probe mix at 90 °C for 10 min and then place on ice.
Excitation
429 ±20 nm
495 ±20 nm
546 ±12 nm
Emission
470 ±30 nm
525 ±30 nm
580 ±30 nm
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