I am staining cells for MAGE-C1 expression but I have experienced autofluorescence of these cells.
The cells are fixed with methanol or 4% Paraformaldehyde or Ethanol.
They are blocked with 5%BSA in PBS or 5%PBS+03% Triton X-100.
What would be the probable cause of the autofluorescence?
Hi Augustine,
I think the autofluorescence is exerted from cell itself after absorbed light (exert from mitochondria, lysosome or ECM...). Trying the use fluorescent dye with different lifetime to distinguish autofluorescence and your interested signal, or trying to decrease the intensity or exposure time of excited laser to minimize the noise signal.
Hope this help.
Augustine
Autofluorescence will always be there to some extent but there are tricks to distinguish it from the true signal. make sure you image your stained cells at the same exposure as your negative controls. Using a non-conjugated prim. antibody and flourescently conjugated secondary can help. Fixation and permeabilization as well as blocking protocols may impact the outcome. depending on where the epitope is you might need to go through additional "unmasking" procedure.
Thank you Anna and Weber. I am investigating whether the target antigen is localized on the surface or Sub-cellular, hence the fixing and permeabilization steps. Also advise if I can be able to stain for the surface and sub-cellular expression at the same and if so, how. Thank you so much.
Hi Augustine,
I suggest that staining your interested protein accompany with marker protein in sub-cellular compartment (e.g. plasma membrane marker or mitochondria marker). The co-localized signal may help you to investigate the precise location of the target protein. And always keep in mind for using distinguishable fluorescent dye for each antibodies.
Best,
I agree, having another target specific for the sub-cellular locus is very important.
you can stain for both at the same time only if the permeabilization of the PM is not affecting the epitope. you have to do some preliminary testing
Is it possible that your problem is the result of low non-specific antibody binding that is not masked by BSA? Try using 5% non immune serum from the second antibody source in PBS to dilute your primary antibody, In addition 0.15 M glycine can be used in your PBS washes to reduce general non-specific background staining. If this doesn't work you can always change the fluorochrome used to alexa fluor 647 and go with a red signal.
Vector Laboratories has released a new product called TrueVIEW Autofluorescence Quenching kit that may help with your application.
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