Hi all
I did transfection in 96 well plates and then fixed it with ice-cold 50%acetone and methanol, but could not proceed with immunofluorescence staining the wells for four days. Would it be possible to visualize the specific fluorescence if I do the staining after four days of fixation??
Hi,
it depends also on your cell type of course....but I have experience on endothelial, macrophages, smooth muscles, and some tumor cell lines and for all of them I saw that waiting more than 3 days after fixation did not give good results.
So I would recommend you to do not surpass the 2 days post-fixation. But this comes only from my personal experience.
Good luck
Any protocol for immunostaining etc are standardized after several experimental trials in order to provide the accurate result. Thus, the established protocol are always used in order to minimize the repetition of the experiments.
Now you have left the micro-plate for more than 4 days in fixative itself, it might have over-fixed the cells or deteriorated/damaged the antigen/protein/epitope of your interest for immunofluorescence staining.
As you know that you need to conduct at least three independent experiments in order to confirm your results. Under this situation, if you try to continue with this micro plate and if you get the expected results but (to be honest) you can not use these data for your scientific interpretation with other two independent experiments wherein you used right protocol and got the results.
Hope, link given below for the methods to perform immunofluorescence staining may be useful:
An introduction to Performing Immunofluorescence Staining. Methods Mol Biol. 2019; 1897: 299–311. doi: 10.1007/978-1-4939-8935-5_26
Chapter An Introduction to Performing Immunofluorescence Staining: M...
Best
Hello Kazi Sarjana Safain
You could have left the fixed cells at 4 degree C for a maximum of 4-6 days if you would have aspirated the fixative solution from the wells and rinsed the cells three times in PBS for 5 mins each, and then stored the fixed cells in PBS at 4 degree C. Cells are allowed to fix in 1:1 acetone/methanol fixative for 5-10 minutes at -20 degree C.
Storing the fixed cells in the fixative solution for days is not recommended as it would damage the cells (antigen of interest) resulting in inaccurate results. It seems that you have stored the fixed cells in the fixative for 4 days.
Best Wishes.
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