Immunocytochemistry for normal bronchoial epithelial cells (ALI culture)

[sunny]

Observed with multiple antibodies (which work great in FFPE tissue) unwanted staining of nuclei when used for FFPE NHBE cells. All, most, or many nuclei show that staining with either DAB or AP detection method. Cells are not very well differentiated in our cultures (when compared to the nice pictures others are publishing of their NHBE cells). Has anybody experienced a similar problem or has anybody an idea why this is happening or how to get rid of that staining?
Thanks,
Sunny

[richard03]

Maybe due to endogenous biotin or antibody concentration too high. Try more diluted antibody.

[sunny]

Thanks Richard for your feedback. We actually use always higher dilutions for ALI culture than for tissue sections because there are so few cells on one slide when compared to tissue sections. When cytoplasm and some nuclei are only counterstained I do not expect that dilution is the problem. We always block endogenous peroxidase, and also used AP instead of DAB detection, but we still have "positive" signals in many nuclei. Could it be that some solutions which are used during culture could have some effect on the staining?

[richard03]

We always block endogenous peroxidase, and also used AP instead of DAB detection, but we still have "positive" signals in many nuclei.

I was referring to endogenous "biotin" not "peroxidase"

Could it be that some solutions which are used during culture could have some effect on the staining?

It is possible. Try to wash more times or use some detergent such as tween-20.

[CanuckPhD]

Have you run a negative control (that is one without antibody)? If it gives you the same result as with antibody then you know you are dealing with something that is endogenous to your system (e.g. biotin).

I would suggest a blocking solution with some Tween-20 (0.05%) to help permeabilize the cells and maybe get rid of any non-specific staining of nuclei. I found this helped a lot when I did cytospins and had nuclear staining.

Good luck

[sunny]

Sorry Richard! Endogenous biotin in nuclei... Never came across something like that in tissues, but actually I just read here http://en.wikipedia.org/wiki/Biotin that "Biotin itself is known to biotinylate histones, but is not found naturally on DNA." I did not know that. I willl check all the solutions used in our cell cultures. Thanks Richard amd CanuckPhD for the suggestion to use Tween 20. I am a big fan of that too and we always used it in the lab where I worked before. However, we used to incubate in a moist chamber and where I work now everybody is using the capillary technique and they say that they can not use Tween 20 because the solution won't be soaked well. But perhaps we could try to block endogenous biotin by incubation with avidin and saturate then all open bindings with a biotin solution before we start the ICC procedure? And we will do one staining without any antibody, one without primary and one without secondary antibody. Thanks so much for your time,
Sunny

[sunny]

Update: Procedure without primary ab resulted in completely negative staining, so it is probably not biotin which causes the problem.

[richard03]

So it is very likely due to primary antibody.

[dutchmaninvienna]

Iīm not a specialist on cell cultures but in archival paraffin embedded tissue often the nuclei start to stain with various antibodies when the tissue hast been too long in formalin. I donīt know how you fix your cells but I would suggest to use freshly prepared paraformaldehyde instead of diluted formalin from the bottle and see if you have less problems when you fix the cells shorter than you are doing now.

[sunny]

Thanks for your replys. Yes, I agree, the problem should be the primary antibodies, if only I knew why this is never happening in the tissues...
Could it have something to do with the differentiation grade of the cells? Did anybody observed that in undifferetiated cells antibodies (which work great in differentiated cells) seem to bind to the nucleus although they should not?
Dutchman, thanks for the suggestion. The problem is, that we are working in these cultures with infectious virus and we have to fix the cells in formalin for a longer time than it would be necessary without the virus to make sure that the virus is not infectious anymore. But thanks again for all you help.

[richard03]

If the antibody worked great in differentiated cells, the nuclear staining pattern may have something to do with the differentiation grade of the cells.

Could it have something to do with the differentiation grade of the cells? Did anybody observed that in undifferetiated cells antibodies (which work great in differentiated cells) seem to bind to the nucleus although they should not?

[CanuckPhD]

I agree that it could be the differentiation stage of your cells. Another thought is how much of a dilution titration did you try for your cells? If you use the same dilution of primary and secondary antibody as for your tissues it may be far too much, which could cause false positive staining.

Good luck