In the procedures of FISH, are non-specific backgrounds (probes) and abnormal DAPI staning caused by :
- Heat Pretreatment ;
- Enzyme digestion ; or
- Other?
* Are these attached images represent problems in previous noticed steps of the protocol?
Thank you.
Dear Mohamed Lahmadi
Can you provide info about the probes used, the cells tested, and the protocol you followed to get these results?
Knowing it might help point to the problem.
Regards
Dear Bassam MS Al-Musawi ,
* These probes (cytocell) for detection of ALK rearrangment in lung cancer tissue (FFPE samples).
I've followed the protocol of "Cytocell" :
- Heat Tissue in pretreatment solution at 95°C, for 15 mins.
- Enzyme digestion for 10min at RT,
- Pre-denaturation : *Add 10µl - 15µl of probe mixture,
Place the slide to pre-warm on a 37ºC for 10 mins,
- Co-denaturation & Hybridization :
Heating the slide on at 75º for 5 min flowing by incubation
at 37ºC overnight (Hybridizer).
- Post-hybridisation washes and with 10µl-15µl of DAPI antifade.
Thank you
Dear Mohamed Lahmadi
Looking at your protocol and from similar results obtained from our experience, it might seem that the enzyme digestion time is longer than required.
Although the manufacturer advised this time, it is sometimes necessary to manipulate the timing or other steps to get satisfactory results.
Try reducing the time for enzyme digestion and see.
Another less likely cause is contamination.
Hope this can solve your problem.
Please, keep us updated, if you may.
Regards
Thank you Bassam MS Al-Musawi;
I will try these recommandations.
Best regards
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