I use a 30bp Alexa labelled probe that binds to expanded repeats. The probe has good foreground signal but also binds to the nucleolus and membranes outside the nucleus and produces lots of background. I tried blocking it in Hyb-buffer (10% dextran sulfate, 50% formamide, 2XSSC, 50mM sodium phosphate buffer, 10 ng/ml tRNA, Salmon sperm DNA, 0.1 mg/ml pH 7.0) for 1hr prior to hybridization. After hybridization I washed the probe (50% formamide 2X SSC) for 3x for 10min each at 55C. I'm still having same background issues, see attached image. Any suggestions would be helpful.
That do look very problematic indead....
How do you visualize the probes? Do you use a fluorophore directly conjugated to the probe, or do you use an antibody system?
Best
Caspar
the probe is conjugated with Alexa dye. i use a confocal microscope to image the foci. the background is from non-specific binding of the the probe.
First suggestion:
Dilute your probe further 1:50 to 1:1000.
Second
Use Prolong Gold antifade reagent to mount the slides.
I think your problem can be resolved.
I usually block in hybe solution for 1.5 hrs not 1 hr at 55C, and after hybridization I perform 4X washes in hybe for 15 min each at 55C. Furthermore, after 4x hybe washes I perform 4x washes in PBT for 5 minutes each at room temperature. I also store and mount my tissue in SlowFade medium. I recommend storing the sample in SlowFade overnight in the freezer before imaging so that the tissue can fully absorb the SlowFade. I should also note I have used this protocol in Drosophila embryos and other Drosophila tissues only.
Hi Sarat, did you manage to reduce your background? I am having similar problems with my FISH experiments in lymphoblast cells.
I could not completely get rid of it but managed to reduce it so that it was not an issue while imaging. After hybridization I increased washing time between 30 to 45min at hybridization temperature and further two washes for 15min at RT. also try adding 0.01% triton to wash buffer it can help reduce background. hope it helps.
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