Immunofluorescence Help

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sar1221's picture
Immunofluorescence Help

 I am trying to perform co-immunofluorescence on HEK293T cells using Alexa Fluor 488 and 594 secondary antibodies.  I have gotten my staining with the 488 secondary to work, but am having problems with the 594 secondary.  When I perform the immunofluorescence procedure without any primary or secondary antibody, I am getting autofluorescence of the cells.  I was wondering if HEK293T cells are known to autofluoresce or if it could be a problem with my immunofluorescence protocol.
yes">  I am fixing my cells with paraformaldehyde and have read that it can cause autofluorescence, but was not sure if it would be a problem with the wavelength that I am looking at.

Arvind Singh Pundir
Arvind Singh Pundir's picture
 check the excitation range

 check the excitation range of 594 in spec sheet and go slight above that and see if your signal is optimised try at around 620