Recommended starting constructs for new DREADD users

For those of you using AAV-based delivery of DREADDs we recommend that you consider using the following constructs/viruses if you are using rodents.

Non-Cre-dependent

AAV8-hSyn-hM3Dq-mCherry
AAV8-hSyn-hM4Di-mCherry


Cre-dependent

AAV8-DIO-hSyn-hM3Dq-mCherry

AAV8-DIO-hSyn-hM4Di-mCherry

We've found these give excellent transduction in a variety of brain regions and high levels of expression in neurons.

Also, if you fix the tissue please consider using an anti-mCherry antibody rather than endogenous mCherry fluorescence to visualize the DREADD-mCherry fusion protein as considerable quenching of fluorescence occurs following fixation.

Bryan

Comments

  1. Immunofluorescence Staining Protocol (mCherry)

    Cryosection:
    1. Perfuse the mice with ice cold 4%PFA/1xPBS
    2. Fix the brain with 4%PFA/1xPBS @ 4°C O/N.
    3. Transfer brain sample to 30% sucrose/1xPBS, allow sample sink @ 4°C for about 48hr.
    4. Crysectioning (40um)

    Staining:
    5. Permeablize the tissue with 1xPBS/0.4% Triton x100 for 1hr at RT.
    6. Block the slide with 1% BSA 5% NGS (normal goat serum) in 1xPBS/0.4% Triton x100 at RT for 1hr.
    7. Incubate the sections with primary antibody (1:1000) in blocking buffer @ 4 O/N.
    8. Wash the section with 1xPBS/0.4% Triton x100 three times at RT, 10min/each.
    9. Incubate the sections with secondary antibody (1:250) in blocking buffer for 2hr at RT.
    10. Wash the sections with 1xPBS/0.4% Triton x100 three times at RT, 10min/each.
    11. Mountain the section with aqueous mounting medium.

    Antibody:
    Primary antibody: Abacm, Mouse anti-RFP monoclonal antibody, Cat # AB65856
    Secondary antibody: Invitrogen, Alexa Fluor® 488 goat anti-rabbit IgG (H+L), Cat# A-11008

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    Replies
    1. Very late to this party...but maybe this will save someone a step. The IF protocol offered by Hu for mCherry works quite well, but the secondary antibody indicated in the above steps is incorrect. You must use a Goat anti-mouse secondary (i.e. Alexa Fluor 488 goat-anti-mouse IgG). Thanks for the protocol!

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  2. Hello,

    I was wondering if:

    1. you can see mCherry without perfusion-fixation?

    2. If perfusion is necessary, would one be able to post-fx the tissue? ie sacrifice mouse, remove and rapidly freeze brain, cryosection, and then fix the sections on the slides?
    If so, would one need to do immuno with an antibody for mCherry as is done when there is perfusion-fixation?

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  3. 1. If you are using non-fixed tissue (e.g. live slices for ephys) you should be able to see the mCherry quite brightly.

    2. Yes you can post-fix--though this usually is not as good as perfusion. Yes I'd recommend using an antibody as fixing usually quenches the fluorescence of fluorescent proteins.

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