Help! Non-specific staining with ICC - (Jul/14/2005 )
I hope you may be able to help me. I'm having trouble with an ICC technique to stain for cell surface antigens. After completing the experiment and trying to view the slides using a confocal microscope I get some surprising results. I have eliminated background staining by using a blocking agent, however, what I'm seeing is what appears to be cell debris on the coverslip. Initially I thought that this may have meant that the cell had burst, however the 'debris' fluoresces at every wavelength on the microscope! This has not been seen by anyone in my lab before, so I need help from elsewhere, and I'm hoping that someone here may be able to help me! Perhaps there is something wrong with my protocol, I just don't know. Protocol I use is as follows
1) Incubate with serum (blocking agent) for 1 hr, then was
2) Incubate with primary AB at room temp for 1 hr, then wash
3) Incubate with secondary AB at room temp for 30 mins, then wash
4) Incubate with texas red at room temp for 15 mins, wash
5) Incubate with acid:alcohol for 2 mins, wash
6) Incubate with Hoesch Blue for 10 seconds, wash
7) Mount and view coverslips
Bit confused! Hope someone can help!
Why do you do an acid alcohol step? I can't imagine this is good for your cells or flurophores? Are you cells fixed? Also could they be bacteria or debris in your mounting medium. Have you examined the slide with and without mountant? Can you post a picture?
The protocol that I am following indicates the use of acidified alcohol to permeabilise the cells in order to counterstain the nucleus with Hoechst blue. Is this step necessary or is there another way, perhaps using a mild detergent? Currently, I do not fix the cells, would you recommend fixing them? and would 4% PFA be ok for this? I would not have thought that the mountant would have bacterial contamination as it has only recently been made up.
Thanx for your help.