Intentional GFP quench prior to immunofluorescence?

Use for discussion of various microscopy and imaging techniques, such as phase, fluorescence and confocal microscopy, electron microscopy, and various other imaging techniques.

Moderator: r.rosati

Intentional GFP quench prior to immunofluorescence?

Postby sdslattery » Feb 11 2009 8:24 pm

Hi,
Is there any way to quench GFP fluorescence in cell to practically zero prior to immunostaining which won't have a negative effect on the immunostaining?

The reason I ask is that I need to perform immunofluorescence on cells and am required to use a green fluorophore. My cells, unfortunately, will be expressing GFP as an shRNA reporter gene, but I'm not interested in that GFP signal, which could interfere with signal quantification of the immunofluroescence.
sdslattery
newcomer
newcomer
 
Posts: 2
Joined: Feb 11 2009 8:15 pm
Location: Baylor College of Medicine

Postby Astarte » Feb 12 2009 8:10 am

You could try photobleaching the cells before immunostaining, but i think they would be recovered by the time your immunostaining is finished. You could look into crystal violet, you should look up if this quenches your fluorophore as well.
Why isn't it possible to use another fluorophore? or use a YFP signal? this would be a lot easier.
Astarte
Prolific Post-Master
Prolific Post-Master
 
Posts: 120
Joined: Nov 13 2007 12:41 pm
Location: Belgium

Postby sdslattery » Feb 12 2009 6:41 pm

In response to astarte:
Thanks for your suggestions.
I think your idea of photobleaching might work, and if it was done after fixation, it would probably stick. But I've never tried it and I'm not sure how much light would be required. I guess it would be easy enough to test, though.
The reason I would like to use a green immunolabel even though my cells express GFP is: I've obtained an shRNA plasmid via a core facility and they will do all sorts of work for me, such as making viruses for transduction, provided I use this plasmid. It expresses the shRNA to my protein of interest but also expresses a GFP reporter for transfection efficiency. While that's nice, my ability to see this GFP reporter isn't really that important. In my assay, I'd really like to be able to stain with four colors: DAPI plus three immunolabels. We're set up to do DAPI plus green, red, and far red fluorophores in the same sample. I don't think with our instrumentation that I can avoid using the green. I suppose I could eliminate the GFP by some cloning, but I'd rather avoid that if there's a simpler way around it.
sdslattery
newcomer
newcomer
 
Posts: 2
Joined: Feb 11 2009 8:15 pm
Location: Baylor College of Medicine

Postby kmunson779 » Feb 12 2009 7:22 pm

In my lab, we've had a much harder time trying to maintain GFP fluorescence than lose it. Our normal fixation protocol (I don't know the details, but it involves paraformaldehyde and acetic acid) doesn't keep the GFP photoactive. We visualize GFP via immunofluorescence with an anti-GFP antibody. If we want to view the GFP directly in a fixed cell, we have to use a different fixation procedure (I think it doesn't use acid, and adds methanol?).

My point is, try fixing a GFP+ sample and look in the green channel. I doubt there will be any signal.
kmunson779
PI of Posters
PI of Posters
 
Posts: 695
Joined: Dec 10 2003 6:23 pm
Location: Ithaca, NY

Postby monicaSE » Mar 03 2009 6:55 pm

Strong reducing agents such as FeSO4 can convert GFP to none-fluorescent form. However it can be restored by exposed to oxygen.
monicaSE
technician-in-training
technician-in-training
 
Posts: 11
Joined: Sep 19 2008 2:16 pm

Re: Intentional GFP quench prior to immunofluorescence?

Postby rnakatsuji » Oct 29 2010 7:48 pm

Hi sdslattery,

I don't want to post any commercially related content, so send me a message or e-mail (rnakatsuji@cri-inc.com). We may have an imaging technique that will solve your problem.

Ross
rnakatsuji
newcomer
newcomer
 
Posts: 1
Joined: Oct 29 2010 7:39 pm

Re:

Postby dutchtech » Feb 11 2011 3:34 pm

kmunson779 wrote:In my lab, we've had a much harder time trying to maintain GFP fluorescence than lose it. Our normal fixation protocol (I don't know the details, but it involves paraformaldehyde and acetic acid) doesn't keep the GFP photoactive. We visualize GFP via immunofluorescence with an anti-GFP antibody. If we want to view the GFP directly in a fixed cell, we have to use a different fixation procedure (I think it doesn't use acid, and adds methanol?).

My point is, try fixing a GFP+ sample and look in the green channel. I doubt there will be any signal.


Don't know what kind of GFP you guys are looking at but in our lab we have no problems at all looking at GFP signal, live or after fixation with 3,7% formaldehyde. Just make sure you keep them out of the light when not working with them.

rnakatsuji wrote:Hi sdslattery,

I don't want to post any commercially related content, so send me a message or e-mail (rnakatsuji@cri-inc.com). We may have an imaging technique that will solve your problem.

Ross


I believe it is ok to post commercial stuff as long as you make clear you work for a company. You kinda made me curious what the technique is :lol:
dutchtech
technophile
technophile
 
Posts: 34
Joined: Dec 01 2009 2:27 pm


Return to Microscopy and Imaging Techniques

Who is online

Users browsing this forum: No registered users and 2 guests