Leica Kreatech FISH probes Mode D'emploi page 14

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As an alternative the following protocols can also be used:
Pretreatment:
Protocol I (standard protocol, if not satisfactory use protocol II)
1. To age the slides, bake the slides for 10 minutes (min) at 80 °C,
then allow slides to cool down to room temperature (RT).
2. Place slides in freshly prepared 70% acetic acid (in deionized
H O(dH O)), incubate for 1 min at RT.
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3. Place slides in 1 x PBS, pH 7.4, incubate for 30 seconds (s) at RT.
4. Place slides in 1 x PBS, pH7.4, incubate for 5 min at RT.
5. Dehydrate slide in 70%, 85% and 100% ethanol, incubate for 1 min
each at RT. Air-dry at RT.
Proceed with Probe
Protocol II (for slides with high cytoplasmic background or direct
preps (amniotic fluid or chronic villi))
1. Pre-warm 2 x SSC, pH 7.0 and 0.01 M HCl at 37 °C
2. Place dry sample slides in pre-warmed 2 x SSC, pH 7.0, incubate
at 37 °C for 2 min.
3. Add pepsin to the pre-warmed 0.01 M HCl to a final concentration
of 0.005%.
4. Place slides in 0.005% pepsin solution in 0.01 M HCl and incubate
at 37 °C for 5 - 15 min (depending on the amount of cytoplasmic
background).
5. Place slides in 1 x PBS, pH 7.4, incubate for 3 min at RT.
6. Post-fix slides by incubating in 1% buffered formaldehyde in
1 x PBS / 20 mM MgCl
7. Place slides in 1 x PBS, pH 7.4, incubate for 3 min at RT.
8. Dehydrate slide in 70%, 85% and 100% ethanol, incubate for 1 min
each at RT. Air-dry at RT.
Proceed with Probe preparation.
preparation.
for 10 min at RT.
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Kreatech FISH probes