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:
- 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.
For visualizing HA we have a detailed recommended protocol the principle of which is below.
I also show various secondary antibodies which might be used along with excitation/emission characteristics
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.
Thanks to abcam for images and spectral properties. We receive no $$ from abcam or any other vendor.