Frequently Asked Questions - Leica Kreatech FISH probes Mode D'emploi

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4. Briefly spin down probe vial, vortex probe vial and then spin down
probe vial again.
5. Apply probe to denatured slide, cover with glass coverslip, seal
with rubber cement and

Frequently asked questions

I have weak or no signals
• Re-hybridize making sure that the probe has been mixed correctly
and that it is at RT before use.
• Re-hybridize making sure that the stringency wash (Wash Buffer I)
is at the right temperature (72 °C (±1 °C)).
• Account for temperature drop when adding slides to pre-warmed
reagents.
• Tissue digestion not optimal (under-digested) – soak off cover
slip in 2 x SSC, pH 7.0 for 2 min at RT. Prolong protein digestion by
2 - 20 min.
• Tissue digestion not optimal (over-digested) – Discard slide and
start with fresh section. Reduce protein digestion by at least
10 min.
• Use a minimum of 10 μl of probe per 22 x 22 mm coverslip.
• Check microscope filters and light source are correct and in full
working order.
I have high background.
• Tissue digestion not optimal. Under-digested tissue increase
background. Soak off cover slip in 2 x SSC, pH 7.0 for 2 min at RT.
Prolong protein digestion by 2 - 20 min.
• Increase the temperature of the stringency wash (Wash Buffer I)
when washing slides.
I have cross-hybridization when using Centromeric probes.
• Increase the temperature of the stringency wash (Wash Buffer I)
when washing slides.
proceed with
Kreatech FISH probes
Hybridization.