3 replies to this topic

[Christina Shi]

Hi,
  Recently, when I do IF experiments, I find GFP tag without any target proteins always locates at centrosome though this kind of dot location is not so obvious, we still can see it. I fix the cells with cold-PFA on the ice for 10min then wash it with PBS 3 times for next step.
  I did the experiments to fix GFP tagged target protein with cold methonal on the ice, but the signal is delocation. It should be a protein that locates on the cenrtrosme. However when I fix the cells with methonal I cannot see any signal in the centrosome. So now I don't know what I can do.
Does any one has any suggestion? Thank you!

Edited by Christina Shi, 24 November 2010 - 05:31 AM.

[Curtis]

both PFA and methanol should work fine. but I have never fixed with pure methanol. it was always 50-50 methanol-aceton. it worked even better than PFA

[Christina Shi]

Curtis, on 24 November 2010 - 06:26 AM, said:

both PFA and methanol should work fine. but I have never fixed with pure methanol. it was always 50-50 methanol-aceton. it worked even better than PFA

Yes,it should be. But the fact seems not so satisfactory. 50-50 methanol-aceton cannot make GFP tagged target protein leave centrosme? I never tried this fixion.

[jonas albarnaz]

Christina,
At present, PFA causes artefacts in some proteins' localization by immunofluorescence. Changing fixation to pure methanol solves the problem.
Best,
Jonas