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
If you have any other suggestion please don't hesitate to tell me, Im looking forward to any information I can get.
wurm11Member Since 18 Mar 2011
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