Staining HT29 cells with TLR Abs and FITC, TRITC and DAPI - (Aug/11/2005 )

Hi all,

I'm trying to stain HT29 cells with primary antibodies NF-KB rabbit, TLR-3 rabbit, TLR-4 mouse, TLR-5 rabbit and secondary antibodies Swine anti-rabbit TRITC and goat anti-mouse FITIC and also staining the nuclei with DAPI. I am using PBS and 0.005% Tween 20 in my washing steps.

I sucessfully completed this staining with CACO-2 cells, I tested out different antibody dilutions (1:200, 1:100 and 1:50) and found that 1:200 gave the sharpest image. However I am not getting any images when I try to do this ecxperiment with HT29 cells even at different dilutions, there is not even any traces of background staining.

I was just wondering if anyone has done any similar staining to this and may be able to shed some light on the problem, It may be the cell walls are very different, but im having difficulties in finding papers about cell lines and their structures.

Any feedback would be much appreciated,

Thank you.

Hi,

not much help to solve your problem, but when I was doing IHC some time ago with HT29, Caco2 and T-84 cells, I did not have to adjust any staining protocols between the different lines.

Sorry, for being more helpful...

PS: I used to use 0.1 % TX-100 PBS instead of Tween-20, but I doubt that this makes the difference

Hey,

Thanks for the feedback, I wouldn't expect there to be that much difference between the cell lines, not the the extent that there wouldn't be any staining at all. I have no idea why its not working as I followed exactly the same protocol! I will try the 0.1 % TX-100 PBS.

thanks.

Are you sure there isn't just different protein expression in the two cell lines to give you a lesser signal in the HT29? This would make sense to me as an explanation.

Is the DAPI as robust in both preps? If not then there is something fundamentally wrong with the prep.

That could be possible, however my IL-8 ELISA results have shown a higher response with HT29 cells compared to the CACO-2 so I would expect the same if not higher levels of protein!

It must have been something I did wrong, But I can't think of what it could be. It is possible to stain for all of these antibodies at the same time isn't it? I didn't try it with as many in the CACO-2 cell lines, but the DAPI response was at the same level.

Thanks

goat-antirabbit secondaries tend to be the best for rabbits so maybe try using that as your secondary. I don't know that it will help but it is worth a shot.

Same time as in on the same section or well?? Don't you have 4 different antibodies? Some are made in the same species right? You can stain multiples at the same time (even if in the same species although it is tricky) but need a different fluorophore for each.

Sorry if I am confused.

Also you comment about whether HT29 and Caco-2 have different cell walls. Well firstly they don't have cell walls at all! Their *cell membranes* may be different but that would only come into play if you do not permeabilize the cells ... do you? Some of your antigens are intracellular and you are going to need to do that. I know you use Tween in your washing steps but it may not be enough.

Make sure to include a positive control, an antibody you know works so to make sure your secondaries are working well. Otherwise I would probably suggest you order new secondaries from a reliable vendor. They aren't that expensive and might be the problem.

Are you sure the signal you got previously was specific? Do you block?

Just some thoughts ...