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I am at the beginning of my PhD and am relatively new to FISH. I have been working with LNCaP cells - a Prostate Cancer cell line. I have had significant protein background however the probes have hybridised successfully.
I am currently trypsinising the cells, hypotonic treatment in 0.075M KCl at 37 degrees for 10 mins (spin at 1,000rpm for 3mins) and then washing 3 times in 3:1 methanol acetone (spin 6,000 rpm for 1 min) and fixing. I then leave to dry overnight and have tried both
- incubating in 2xSSC for 10 mins at room temperature, washing with PBS and incubating at 0.001% pepsin in PBS for 10 mins at 37 degrees
- incubating in 2xSSC, 0.001M HCl and 0.001% pepsin
Then washing in PBS and dehydrating in ethanol before probe hybridisation.
Does anyone have experience of working with LNCaPs or cancer cell lines? Should I try using 0.005% pepsin at 0.01M HCl? Any ideas?
Thanks very much
Wed Jan 26, 2011 11:19 am
gula
Rank: Member
Joined: Mar 20, 2008
Posts: 94
Post subject:
I can't help with your special test, but we once had problems with high background when using charched slides (superfrost plus). It turned out, that the background was gone after heating the slides for half an hour in the 60°C dryer instead of air-drying.
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