I am looking to exclusively stain the cell membrane on the outside, no inner membranes post fixation/permeabilization to have an indicator if my protein of interest is on the cell surface or only intracellular.
For this i am looking for a marker/ or general staining that can be mulitplexed alongside DAPI, Alexa Fluor 488 and Alexa Fluor 546.
I looked at multiple staing kits especially for lipid stainings, but have so far not found something that can be used on fixed cells that also is stable throughout permeabilization.
Thank you for the input :)
Article Preclinical evaluation of a TEX101 protein ELISA test for th...
Hi Henrik,
you could try one of the FM 4-64 dyes. I use them to stain the outer membrane of cells in vivo and isolated organelles in vitro, and they work really well. There are also some versions of it that are fixable with PFA or glutar aldehyde (the FX versions).
https://www.thermofisher.com/order/catalog/product/T13320
I hope this is of any help :)
I'd recommend staining the extracellular protein without a permeabilization. You can fix the cells and just add block to the cells/tissue (serum of same species as secondaries or 3% BSA). If you also have an intracellular target, you can permeabilize after the extracellular stain has been performed (just one extra step). This staining procedure is something I have tried and seems to work well, and avoids intracellular staining for an extracellular target.
Hi Elliot.
I wanna stain early endosomes in mice dendritic cells and the secondary antibody I had used is AF 488. but I found very very low signals of eea1. when did the same experiment with AF- 594 signals are good enough. what could be the possible reasons. Can you please let me know.
thank you
@madeeha-- could be a bad secondary antibody or a laser issue on the 488 laser. If you are getting good signal with your primary + secondary and no signal with a secondary- only control, you should feel confident with your result. Good luck!
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