I am struggling with detecting 17p deletion in Chronic lymphocytic leukemia (CLL) by FISH - IS using BAC ( bacterial artificial chromosome) with ImageStream X (Amnis), otherwise centro-mere probes works well.
Many thanks,
Cheers,
Cuc
Hello Cuc Hoang Do
you have to test the BAC on the normal human chromosome to see the signal
I have done FISH on normal male chromosome to check our BAC probes. Every BAC clone probe hybridize at appropriate location with good signal.
Many thanks for your question.
Did you try pretreatment of the CLL-slides? Pepsin treatment may be helpful to enable the probes to get through the proteins of the preparation.
I have done pepsin treatment on CLL cells in suspension before hybridization with probes, but cells are distorted. Could you please suggest a protocol using pepsin pre-treatment? Thanks.
you could do a prefixation step before the pepsin - i.e. rinse slides for 3% in (para)formaldehyde solution.
Thanks for your advice.
I should be clearer. I have fixed CLL cells with Carnoy's solution( Methanol:Acid Acetic 3:1) before pepsin treatment but they 're still distorted. Could you please show me your protocol if possible? I am wondering the concentration of pepsin using?
after dropping the slide let it dry overnight and then put it for 2-5 min a 3% (para)formaldehyde solution. Then cells are stabilized and the you may use normal pepsin treatment and cells should be ok.
Also it happens when you change the batch of pepsin that you have to test for concentration and time of treatment
- Badges
- Science topic