I'm picking up an old project started before I joined the lab...My lab has cells that were fixed in 10% formalin in March 2020 (almost three years ago). Due to the pandemic, the cells were never stained. The cells were kept in PBS at 4C. I have never stained cells that long after fixation; I usually fix and stain the same day. So I'm wondering if the cells are still usable. How long will formalin-fixed cells be "good" for immunofluorescent staining? Thanks!
Well, crosslinking with formaldehyde is covalent, though there is the potential that any proteases in the solution could cause gradual breakdown of cellular proteins. The bigger worry is that storage in 4C will not prevent bacterial or fungal growth. Even addition of sodium azide may not be sufficient after all of that time. So you may have contamination as well as pH imbalance of the buffer. There's also the concern that the water in the buffer could have evaporated to some extent over time, leading to salt crystal formation and pH imbalance.
Instead of 4C in PBS, I would have recommended storage at -20 in a cryopreservative. But in this case, of course, it's too late.
My recommendation would be to closely examine the cells under a microscope to see if there is any bacterial/fungal growth. If not, then I'd remove a sample, rinse it well in fresh PBS, and give it a test stain using a validated primary/secondary.
Hi Hannah Youngblood
I agree with Jason A Kilgore
In addition to that, I observed the degradation of cells when I kept formaldehyde fixed cells for 1 month, that is maybe because of bacterial/fungal growth. So, its better do the staining within the week.
Thanks
Pankaj
Hi Hannah Youngblood
I have usually have tried keeping it for 4-5 days and stained, it works fine. However on prolonged storage for more than a week the cells start to die and eventually the number of cells decrease.
* I fix the cells in 4% PFA (10mins) and then add PBS and store it at 4 degree.
I have done the same with lots of different cell lines and did not find any alterations in results.
Hope it helps
Thanks
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