I'm running Immunofluorescence imaging today, and I added the antibody. Cells are i at 37 incubator for four hours. I wont be able to finish imaging today due to the number of samples. Can I leave my cells for four days at fridge in the diluted antibody to continue imaging later? If I remove the antibody and add PBS, can I detect any signal after four days?
Thanks
Hi, I suggest you to do the staining, then remove the antibody and after that fix. IF should last more than 4 days. I have done some flow cytometry after 3-4 days and still looks great. Although I advise you to read about the molecule you are trying to detect. Sometimes long periods of fixation affect the structure and fluorescence.
Do not let the cells incubate with the primary antibody for so long. The longer it takes, the more background you get. Its better to leave them in PBS solution.
As others have said, prolonged staining increases the background with no further increase in signal. Likewise, leaving stained tissue in PBS-T will deplete signal intensity over time. If you need to wait before staining, samples can be stored in 70% EtOH before starting staining, or can be stored in a glycerol based mounting media at 4oC or even -20oC.
Thank you all for your advice, I stayed till after midnight and finished the imaging!
I checked the cells after four days and couldn't detect any signal!
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