Dear RGfriends, I would like to stain primary cells against Ki67-antibody (and more antibodies) at different cell passage (from P=1 to P=6). In order to run the ICC for all samples at the same time, I thought to fix the cells with 4%PFA (10min) and then store them in PBS at 4 degrees until I will collect all samples. Would it be ok? Thank you in advance for your suggestions and help. Looking forward to hearing from you in the near future and in the meantime I wish you a great weekend. Best Regards,
Mario
You can fix the cells in 4%PFA/PBS and after washing them 2x in PBS, you can leave the fixed cells in PBS at 4*C for not more than 10 days.
Dear Milica,
many thanks for your answer. The problem is that in order to collect all Passages I have to wait for more than 1month. Would you suggest an alternative way? I thought also to freeze an aliquot of cells for each cell passage and then seed all of them in the same time. By using this way, probably I need to leave cells to grow and stabilize at least for one passage and then use them for ICC in the next passage, losing a cell passage and affecting further the molecular biology background of those cells. This is my thought. Or, would you suggest to thaw the cells and use them straight away
for ICC? Many thanks again for your tip. Have a great weekend, Mario
In my experience, PFA-fixed cells retain their ultrastractural features also for 2 months if fixed for 20 min and then stored at 4 C in PBS with 0.05 % sodium azide. By the way, you should compare your staining at 1 d and 30 d post-fixation, in order to exclude the presence of any artifact.
Ciao Sergio. Thanks for your comment. Well, the cells of last passage would be stained after 10-20min PFA-fixation, but they would be not a good comparison since the cell passage will be different and I expect a ki67 expression changing by cell passage manner. I do not know how to make the comparison you suggested. Also, would 20min PFA fixation be better than 10min always, even if I will stain cells straight away? Thanks again for your suggestions. Have a great weekend. Best Regards, Mario
Dear Mario,
If I understood your problem, you can fix n samples of your cells at the same passage, then you will stain fixed cells after different time post-fixation (i.e., 1 d, 2 weeks, 1 month, etc.). What do you think?
Have a good time
Sergio
Hi Mario,
After fixing your cells, instead of leaving them in PBS at 4*C, aspirate PBS, dry the cover slip and freeze cover slips with cells at -20*C. In this condition, you can keep cells for a long time until you finally finish with collection of all passages.
Probably you already grow them on cover slips or similar, but the point was that you can store them at -20*C for a longer period. Once you need to do the staining, take the cover slips on the room temperature, wash with PBS and continue with staining.
Best.
Milica
Milica Bozic
Dear Milica,
do you know for how long can the dry cover slips be stored at -20C without losing their integrity?
Do you have any literature recommendations to look for?
Thank you and best regards,
Thanos
Dear Athanasios,
Unfortunately, I do not have any literature recommendations regarding this issue. Generally, I always try to do immunostainings from freshly grown and fixed cells or at least cells that did not stay longer than 2 weeks in the fridge after fixation. For some experiments, I stored cover slips for up to 3 or even 6 months at -20°C (after fixation) and I was able to detect the protein of interest later. Nevertheless, everything depends of the protein and its abundance in the cell, localization, stability, etc … So, you need to be careful with this and to test the best conditions for your protein.
Best of luck,
Milica
Yes, it is possible to store 4% PFA fixed cells in PBS at 4 degrees for many days. However, the length of time that the cells can be stored in this way depends on the specific cell type and their environment. It is recommended that the cells be monitored regularly for any signs of degradation.
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