Considerations for immunofluorescence for various DREADD constructs

I receive frequent queries regarding the appropriate way to visualize DREADDs.  Below I show the three basic DREADD constructs:

  • HA-tagged
  • mCherry fusion proteins
  • HA-tagged in a vector with IRES-mCitrene
In unfixed tissue the mCherry and mCitrene can be visualized using the parameters from the table below or by going here.

NOTE: only the mCherry fusion protein fluorescence will show that the DREADD receptor is expressed.  mCitrene simply will show which cells were transfected or transduced.





































Here's what you might consider if you have an mCherry fusion protein:




Finally, what if you are using one of the HA-hM3Dq-IRES-mCitrene or HA-hM4Di-IRES-mCitrene constructs.  Here you would want to use our HA staining protocol and then use an anti-mCitrene primary/secondary combination (one is shown below) so that the HA-tagged DREADD is visualized in the RED channel and the mCitrene in the GREEN channel.





Acknowledgements: 
Thanks to abcam for images and spectral properties.  We receive no $$ from abcam or any other vendor.





Comments

  1. Hi Bryan,

    I see above that you recommend the use of anti-Citrene antibody. Is the Citrine/Citrene used in the AAV-CaMKIIa-HA-hM3D(Gq)-IRES-mCitrine construct detectable by common anit-GFP antibodies? In general I thought that anti-GFP antibodies detect GFP, CFP, YFP and even Citrine. If not do you have a recommended anti-Citrine/Citrene antibody?

    Cheers

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  3. Polyclonal antibody can detect mCitrine. We are using the rabbit anti-GFP polyclonal antibody from Novusbio, NB600-308 (1:1000). It works great.

    Best,
    Hu

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  4. Hi, Bryan,
    Do you have any recommendation on mcherry antibody? Which brand is better?

    Thanks.

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  5. Primary antibody: Abacm, Mouse anti-RFP monoclonal antibody, Cat # AB65856
    Secondary antibody: Alexa Fluor® 594 Goat Anti-Mouse IgG (H+L) Antibody, Invitrogen, Cat# A-11005

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  6. Hi,
    are there evidences about a preferential localization of DREADDs in neurons, please? (axon terminals, somato-dendrites..)
    Thank you in advance.

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  7. There is no information though hM4D tends to be slightly axonally targeted

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  8. Hi,
    Unfortunately, Abcam is no longer producing Mouse anti-RFP monoclonal antibody, Cat # AB65856. Is there any equivalently working antibody on the market that you can recommend, please?
    Thank you very much.
    -Christiane.

    ReplyDelete

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