Leica Kreatech FISH probes Mode D'emploi page 16

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4. Place up to 14 slides in 200 ml of pre-warmed Wash Buffer I (0.4
x SSC / 0.3% Igepal) (LK-102A), incubate for 2 min at 72 °C (±1 °C)
without agitation. Re-use only once for a total of 28 slides.
5. Place up to 14 slides in 200 ml of fresh Wash Buffer II (2 x SSC /
0.1% Igepal) (LK-103A), incubate for 1 min at RT without agitation.
Re-use only once for a total of 28 slides.
6. Dehydrate slides in 70%, 85% and 100% ethanol, incubate for
1 min each at RT. Air-dry at RT.
Proceed to Counterstaining.
Counterstaining:
Apply 15 μl DAPI counterstain (LK-095A (0.1 μg/ml) or
LK-096A (1 μg/ml)) and apply glass coverslip. DAPI can be diluted in
counterstain diluent (LK-097A) to obtain desired concentration. Place
slides in the dark and allow 10 - 15 min for counterstain to develop.
Proceed with microscopy.
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
Excitation
429 ±20 nm
495 ±20 nm
546 ±12 nm
Kreatech FISH probes
Emission
470 ±30 nm
525 ±30 nm
580 ±30 nm