How to preserve GFP signal?

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How to preserve GFP signal?

Postby Manuora » Jul 31 2012 8:30 am

Hello all,

I'm trying to knock down a target gene in vivo by stereotaxic injection of lentiviral vector in rat brains. I use a emGFP reporter under CMV promoter control.
After perfusion with PFA 4%, I cryo-section the brain and look at the fluorescent signal of GFP to validate the effective transduction of neurons.
However I often loose the GFP signal, particularly at the injection site. I just can observe a very low signal along the way of the needle and at the surface.
I have tried to immunostain the sections with anti-GFP antibodies but it never worked and after treatment, the signal is exactly the same.

Does anybody have an idea about what could quench the GFP signal or denaturing the protein?
Thank you for any kind of help.

Manu
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Re: How to preserve GFP signal?

Postby CrowSan » Aug 06 2012 9:34 am

Is it possible that the problem is not with the GFP signal being denatured/destroyed but instead is because the lentivirus in not expressing/integrating in the rat brains? Perhaps the needle is causing damage and the neurons in close proximity to the lentivirus injection just apoptose and die? Or perhaps the lentivirus is killing them. Or perhaps it is not transfecting them.

Do you have a control of some type? Does the retrovirus give a signal in neuronal cells in culture (in vitro)?
As it's brain I am assuming you are cutting frozen sections and not embedding in wax etc.
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Re: How to preserve GFP signal?

Postby LeserattePD » Aug 16 2012 4:26 am

My suggestion would be to get a commerically available GFP expressing mouse as a control to test whether it is your fixation procedure that is the issue or non-expression from the vector as Crowsan suggested.

Yes, GFP signal may be destoyed by fixation, but normally this can be rescued by using an antibody for staining.
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Re: How to preserve GFP signal?

Postby CrowSan » Aug 16 2012 10:16 am

Depending upon the fixation type you may also have to antigen retrieve the tissue to get a good signal (I had to do this to get a good signal with a collagen antibody on parafin fixed sections). A good control will give an idea of which step is going wrong.
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