Hi Melanie, is your primary labeled with alexa fluor 488? Is it a monoclonal?

If so, you should get a hold of polyclonal secondary Abs with red emitting fluors.

Usually secondary polyclonal Abs labeled with red emitting fluors, like  Texas Red, Cy5, etc., have less background issues. Plus,weak green emitting dyes usually do not bleed strongly into the red and infra-red channels.

Polyclonal antibodies made against monoclonal Abs have multiple binding sites, so the alexa fluor tags on your Ab  should not be a problem.

The fluorochrome may prevent a secondary antibody from binding to your primary antibody due to steric hinderance.  I don't know how big the alexa dyes are.  You could try it and see.  Some companies will provide free samples of antibodies for you to try.  You may be able to find an antibody that will bind to the alexafluor tag.  This would be a better option if one is available.  Yes, the alexa conjugate will still emit a signal, so you should select a different fluorochrome.     

hi Melanie, 

why not try to enhance the signal just use the secondary alexa fluor, but the same type of alexa fluor that has been conjugated with primary antibodies -

good luck

Tatyana

Another option would be, of course, to use an unlabeled primary antibody and an Alexa secondary antibody, this way you would avoid the steric hindrance and the residual fluorescence problem altogether.

I also like Tatyana suggestion, in case you need to use slides that you have already stained with your primary.

Thanks everyone. The primary we have is a polyclonal AF488. The AF molecules are pretty small compared to the size of the antibody. AF488 has a MW of 643 while an antibody MW is ~150,000. 

Mainly I wanted to see if anyone had done this before. Currently we have this direct labeled primary and an unlabeled primary (that we've used with HRP secondary for IHC) that we could use for our target, but thought it might lead to increased fluorescent signal if we used the labeled primary, holding any possible issues with a secondary not binding or covering up the primary fluorescence. 

Also, ultimately we want to do a double stain which is why we're still working in the green channel as well as to stay within the filter set we have on our microscope, but if we can't get some excellent signal to background we'll have to switch back to enzymatic! 

Hi Melanie, did you buy the labeled poly or did you do the AF488 labeling in house?

Labeling Abs can be a big headache.

Anyway, I (and probably many others) have had to use labeled secondary Abs to augment the signal of a labeled primary Ab just to see if the primary worked. I have never published any of those results.

That is why you will probably not find any peer-reviewed publication that describes using a labeled Ab to increase the signal of another labeled Ab. This will be a HUGE red flag to reviewers.  

Steingrimur,

We purchased it already labeled. 

That would make sense why I've never seen it then! What exactly makes it such a huge red flag to reviewers? Just because it's not a legitimate method used for signal amplification?

Hi Melanie, reviewers will see that you are not getting a good signal using your primary labeled Ab.

That usually means that your antigen is not abundant enough to be well detected by your primary. So the question will be; if you are not getting a good signal with your labeled primary, then why are you adding a labeled sec Ab?  

Since your primary Ab is a poly, then that opens up a plethora of questions:

-Is the poly affinity purified?

-What else does your poly react to?

-What exactly are you amplifying? etc, etc

It will be a lot less of a problem if you first use unlabeled primary and then come in with a labeled sec. Then the controls will be simple.

BTW, if you are having problems with autofluorescence, this technique might come in handy:

http://www.uab.edu/highresolutionimaging/autofluorescence

The way how secondary antibody could applied is in attachement.

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