Leica Kreatech FISH probes Mode D'emploi page 6

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As an alternative the following protocols can also be used:
Slide pretreatment
1. Mount 4 - 6 μm formalin-fixed paraffin-embedded (FFPE) tissue
sections on positively charged slides.
2. Bake mounted FFPE tissue sections for 2 hours at 80 °C or 16
hours at 56 °C.
3. De-paraffinize slides in xylene or xylene substitute, incubate for
2 x 10 minutes (min) at room temperature (RT).
4. Re-hydrate slides in 100%, 85% and 70% ethanol, incubate for
3 min each at RT.
5. Place slides in deionized H
Proceed with Protocol I or Protocol II.
Protocol I (standard protocol, if not satisfactory use protocol II)
1. Pre-warm 0.01 M sodium citrate pH 6.0 to 96 - 98 °C.
2. Pre-warm 0.01 M HCl to 37 °C (if not using Ready-to-Use (RtU)
pepsin (LK-101A)).
3. Place slides in 0.01 M sodium citrate pH 6.0 at 96 - 98 °C, incubate
for 15 min.
4. Place slides in dH
5. Add pepsin to pre-warmed 0.01 M HCl to reach a final
concentration of 0.025%. (if not using RtU pepsin (LK-101A)).
6. Digest slides in 0.025% pepsin in 0.01 M HCl at 37 °C or cover
tissue in RtU pepsin (LK-101A) at RT, incubate for 5 - 45 min, (time
depending on tissue fixation and tissue type).
7. Place slides in dH
8. Place slides in 1 x PBS, pH 7.4 or 2 x SSC, pH 7.0, incubate for 5 min
at RT.
9. Dehydrate slides in 70%, 85%, and 100% ethanol, incubate for
1 min each at RT. Air-dry at RT.
Proceed with Probe preparation.
O (dH
2
O, incubate for 2 min at RT.
2
O, incubate for 1 min at RT.
2
O), incubate for 3 min at RT.
2
Kreatech FISH probes