High centrifugal speed, elevated temperature when using hypotonic buffer and prolonged reaction time are all possible causes for this phenomenon. Please modify your experimental conditions accordingly.

 To resolve the problem of excessive cell debris, we recommend that you wash the cells with a fixing solution multiple times to obtain a clean cell suspension before preparing the slides, especially for lymphocytes cultured from whole blood. Despite some inevitable cell loss due to the washing process, cell debris will also be eliminated. In principle, enzyme digestion treatment can be omitted when using chromosome slides or cell slides for the FISH experiment; however, if there is excessive residual cytoplasm in the background, you can remove it with enzyme digestion treatment.

 We do not recommend that you use different probes on the same sample slide. Although most probes can be removed by denaturing solution at high temperature, a small amount of probes may still remain on the slide and result in background interference. However, the same probe kit can be used repeatedly on the same spot for FISH experiment.

 The frequency depends on the usage. However, we recommend changing no less than once a week.

 Wash the slides with xylene for 2 minutes to remove the immersion oil. Rinse the slide with methanol for 2 minutes. De-stain the slides with fixative (methanol: acidic acid, 3:1) for 20 minutes. Rinse the slides in H₂O twice for 5 min each. Incubate the slides in 1X PBS twice for 5 min each. Dehydrate slide in 70%, 90%, 100% ethanol, 1 min each. Air-dry the slides.

 Longer denaturing time or higher denaturing temperature may be required for over-aged slides in order achieve adequate denaturing of the chromosomes.

 The metaphase spread slides can be kept in dry containers at -20°C for approximately six months; avoid moistening, e.g. through repeated freeze/thawing. Slides can also be stored at -20°C in 100% ethanol for several years. We recommend that, prior to use, to pre-warm the slides at 37°C for 30 minutes to remove any moisture.

 Slides should be aged at 37°C for 1-3 days before use. For rendering chromosome structure resistant to the subsequent DNA denaturing, slides can also be aged overnight at 65°C or incubated at 90°C for 30 minutes. The slides then can be stored at room temperature for a few weeks.

 Metaphase cell preparations can be stored in fixative solution (methanol: acetic acid, 3:1) at -20°C for several years.

 The cells should be fixed thoroughly with fresh fixative and washed several times to remove the cytoplasmic residual that may interfere with the hybridization procedures. Please refer to Technical Tips.