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Author Topic: Subtype specific antibodies and immunofluorescence staining  (Read 3509 times)

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Offline SSpeese

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Subtype specific antibodies and immunofluorescence staining
« on: December 03, 2013, 01:11:09 AM »
I am curious to get readers input on some fluorescent antibody staining on whole mount tissue we have recently seen in our lab. 

Our first issue is with using IgG secondaries from Jackson with differing subtype primaries.  We have this issue where some users in lab have been using a Jackson IgG Alexa 647 secondary to recognize an IgG2a primary and they are getting very poor staining.  However, if they use a Jackson IgG Rhodamine Red X secondary to stain for the same IgG2a primary, they get very good staining that is eliminated in an animal that is null for the protein the IgG2a primary recognizes, and thus is specific staining. You might think the solution is easy, the Jackson IgG Alexa 647 secondary has gone bad.  However, if I use that same secondary to stain for an IgG1 primary, I get great staining.  Both secondaries are from Jackson and both are Donkey anti-mouse IgG which have been cross absorbed against the same species. Any thoughts ?

The second issue has to do with using subtype specific antibodies from Jackson. We always seem to get better staining with mouse IgG secondaries than when we use subtype specific secondaries to recognize the same primary. Our first though was that at the same concentration the IgG secondary would have less antibodies that would recognize our primary, as compared to the subtype specific secondary.  Thus, we thought we might need to dilute the subtype specific more to avoid background issues and still achieve staining as robust as the IgG secondary.  However, this did not work and when we called Jackson technical support they told us the complete opposite and that we needed to use the subtype specific antibody more concentrated than if using an IgG secondary. Their explanation of why this is the case was not clear so I am curious if anyone else wants to take a shot at explaining this ?

I would also like to know if there are any important considerations when using an IgG3 primary, as far as selection of secondaries.

Thanks,
   Sean Speese

Research Assistant Professor
OHSU-Jungers Center for Neuroscience Research 

Subtype specific antibodies and immunofluorescence staining
« on: December 03, 2013, 01:11:09 AM »

Offline ImmunoNYC

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Re: Subtype specific antibodies and immunofluorescence staining
« Reply #1 on: April 18, 2014, 09:13:03 AM »
How are you imaging the AlexaFluor647 staining? Confocal?

I suggest you contact Jackson IR directly again and specifically ask the questions you are asking - for example why is the subtype specific secondary less robust than the whole IgG secondary? They have an enormous amount of experience and detailed lot information and characterization on their antibodies which may help understand the difference.

I consider each and every antibody different, especially polyclonals and they all need to be titrated independently. There is no one size fits all IHC protocol. I don't assume just b/c, for example, 1ug/ml is optimal for one anti-mouse IgG-AlexaFluor488 it will also be optimal for anti-mouse IgG-Rhodamine. Each antibody would have a different fluorophore with different excitation and emission characteristics, and each antibody may have a difference range of fluorophores per antibody - or even potentially be conjugated with a different technique. Again, Jackson IR should be able to shed light on all of this.

Re: Subtype specific antibodies and immunofluorescence staining
« Reply #1 on: April 18, 2014, 09:13:03 AM »