I am using chilled methanol:chloroform (60:40) for 2-5minutes at 4degree. But the cytoplasm is getting damaged and while using 4% PFA for 30 minutes the nucleus in not conspicuous. Please suggest me.
Thanks
I used 2% PFA before and I did not have any problem in IFA analysis. You can try this.
Good luck
Fixation with 3.7% PFA for 15 minutes yielded good results in our case.
I used 2% PFA 15 min to fix PC-3 cells and I obtained good results too.
Good luck
Dear Guru,
I agree with all comments above from Carole, Moumita and Aysegul. I do not understand what do you mean when you say "while using 4% PFA for 30 minutes the nucleus in not conspicuous". "Conspicuous"? you mean you can not see a obvious nucleus before or after any staining?
I believe, in fixed cells, not seeing nucleus is a good sign. Usually an obvious nucleus is a bad sign indicating swollen or hydrated nucleus with unhealthy nuclear and cytoplasmic membrane. To me sounds like you have very good and healthy cells following PFA fixation. If I were I would stick to PFA in guidance of above comments (15-20 minutes in 4% PFA).
best wishes,
Refik
Thanks everyone for the useful comments.. and Dear Dr. Refik, i meant that while using 4% PFA for 30 minutes, DAPI staining is good but the DIC image does not show defined nuclear wall. Rest is ok.
Dear Guru,
DAPI usually gives a diffuse labelling of the nucleus, and does not give a well-defined nuclear wall in my healthy neurons either. I am not sure if that is due to PFA fixation? You may need to use more specific labels for the nuclear membrane.
best wishes,
Refik
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