I am doing immunostaining of microtissues for collagen I, actin ,ad nuclei.
I already did the secondary antibody step and washed with TBS. Something came up and I won't be able to continue the other steps of the immunostaining today. These steps include permeabilize, blocking again and staining actin and nuclei. Is it ok that I leave my samples in TBS at 4 C and continue after two days?
Most likely, it should be OK, cells may get damaged sitting in aqueous medium so long. But for publication, I will follow the proper protocol. You can not write in procedure, my procedure worked even 2 days after the last step of staining,
I think the samples are unusable, sorry.
If your cells were not fixed, the TBS buffer will screw up cell structures because of salt and protein diffusion going on from the dead cells to the outside, even at 4oC.
If the cells are fixed using aldehydes (formalin, etc) you are still screwed because the tris base in TBS can undo aldehyde crosslinks.
I would start afresh because, if nothing else, the Materials and Methods section would be easier to write.
It should be fine if they are stored cold and in a neutral buffer. Initial fixation preserves all your epitopes, and the binding of primary and secondary to their antigens is essentially irreversible. So the antibodies that bind won't wash out, and you can go back and apply your counter-stains. Good luck with your experiment.
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