4 replies to this topic

[wurm11]

Hi everyone,

I have an urgent question and I hope that someone can help me.

I am currently trying to do Immuncytochemistry with U87 cells (Glioblastoma derived cells) that were stably transfected with a Flag-taged construct.
With Western Blot experiments I could previously show that my Flag-taged constructs are there. I get nice and thick bands and none in untransfected U87 cells (negative control).
The problem is that in ICC I see the same for untransfected cells as for transfected cells with my Flag-taged construct. It seems to me that Staining in U87 with Flag is not working at all because you can not distinguish between positive and negative controls. Im confused as my WB worked well.

I also read that staining with Flag-Ab (monoclonal, F2555 from Sigma) always gives a high background but I couldn't find anything that untransfected cells also give positive signal. Background would not be the problem. Something else is wrong and I can't figure it out as I'm not experienced in that field at all.

Hope I was clear anyway...and I'm looking forward to hearing from you!

Cheers!

[wurm11]

Hi everyone,

I have an urgent question and I hope that someone can help me.

I am currently trying to do Immuncytochemistry with U87 cells (Glioblastoma derived cells) that were stably transfected with a Flag-taged construct.
With Western Blot experiments I could previously show that my Flag-taged constructs are there. I get nice and thick bands and none in untransfected U87 cells (negative control).
The problem is that in ICC I see the same for untransfected cells as for transfected cells with my Flag-taged construct. It seems to me that Staining in U87 with Flag is not working at all because you can not distinguish between positive and negative controls. Im confused as my WB worked well.

I also read that staining with Flag-Ab (monoclonal, F2555 from Sigma) always gives a high background but I couldn't find anything that untransfected cells also give positive signal. Background would not be the problem. Something else is wrong and I can't figure it out as I'm not experienced in that field at all.

Hope I was clear anyway...and I'm looking forward to hearing from you!

Cheers!

[bob1]

Do you have another plasmid -that others have used in ICC- that you could use as a positive control for flag transfection?

What do your antibody controls look like for the flag staining?

Background staining will happen in both transfected and untransfected cells - it is non-specific binding of the antibody to cellular components.

Could you post your fixing and staining protocol so that we can see if there is some methodological error?

I do get some background when staining with flag, but usually the positive signal is quite strong, so the background isn't a problem.

[wurm11]

Unfortunatelly I don't have any other positive control for Flag as I am the first one to establish Flag in ICC.
Background staining wouldn't be the problem if I could clearly distinguish between transfected and untransfected cells but i can't.
Do you also use glioma derived cell lines? I heard that Flag is especially causing a problem in brain cells .

So far I used this staining protocol:

- Fixation with 4% PFA, 15min, RT
- Permeabilisation with 0,3% Triton in PBS, 15min, RT
- 2x wash with PBS
- Blocking with 5% NGS in PBS
- Incubation with monoclonal mouse anti-Flag M2, 1h, RT
- 3x wash with PBS, each wash 5min
- Incubation with secondary AB goat anti-mouse Alexa 488 or 568 (488 in my hands gives a stronger but nevertheless not more specific signal)
- DAPI 1:1000, 15min, RT
- mounting


If you have any other suggestion please don't hesitate to tell me, Im looking forward to any information I can get.
Thanks!
-

[bob1]

I would wash a lot more after the fixing - PFA wil give you quite a bit of background unless the free-aldehydes are washed away.  I would also wash 3 x after the secondary step, and I would make all antibody washes more stringent by using 0.1% tween in PBS (PBS-T).