In my experience Yes. I had a similar incident a few years ago and had huge amount of background staining and unspecified binding.

If you have option to redo with a clean one, you should. If you dont have that option and you HAVE to use the same then, I would check what kind of bacteria they are and see if the specific antibody binds to the surface proteins of that bacteria, If they do then you are out of luck.

Thank you. We're repeating with the clean one.

So it depends on particular antibody. I was wondering if there are some mechanisms of innate immune system, targeting all bacteria, that still can function in vitro in a system that has bacteria, antibodies and serum of different species.

You could apply a drop of blocking solution to a coverslide, let it dry so it sticks to the glass and add your antibodies to see if you get staining. Bacteria are often "sticky" and bind to any kind of proteins, also antibodies and cells. If you ever tried to stain a contaminated cell culture, you know that you get signal everywhere.

Could you describe which antibodies you are using? Commercially available antibodies should be purified to apoint that you don't get any contaminating signals from non-specific antibodies. Otherwise any staining you perform bears a risk of background that is difficult to explain.

Good luck!

We used commercially available antibodies: mouse anti-SSEA4 antibodies conjugated with fluorophore and combination of primary unconjugated rabbit anti-cytokeratin and conjugated secondary goat anti-rabbit. The samples were fixed mouse cell cultures, the blocking solution contained non-fat milk, BSA and normal horse serum. The serum is luquid and contains sodium azide, other components of blocking solution are stored as dry powders. Milk is from grocery shop and looks really old though.

Thanks for the details!

The datasheet for your antibodies should contain some information about how they were purified and how pure they are. But I am pretty sure they don't kontain complement or other contaminationg antibodies.

I know that every lab has their own philosophy about blocking solutions and I have tried many myself. What I figured out was, the simple thing is often the best. Try only BSA, that should block well enough for immunofluorescence; and keep the old milk for western blotting. BSA can be stored frozen in aloquots and then you don't need azide either. You will save your PI a lot of money and reduce the health risk. But if you really have azide in your solution (and it is protected from light so the azide does not degrade), you can be pretty sure that you don't have bacteria growing in the blocking solution.

If you have to continue with your blocking solution, I suggest sterile filtration. You keep the blocking proteins, but remove possible bacteria growing in the old milk powder ;)

Best of luck!

Thank you Tilo for the detailed answer!

In this lab I'm only a trainee so I'm not really allowed to change their practice, like what protein use for blocking, but I can add some steps like filtration to be more accurate.

And your advice will definitely help me in the future!