Re: GFP tissue preparation for confocal microscopy

Posted by mmodel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-tissue-preparation-for-confocal-microscopy-tp2680696p2682906.html

Young - We have a (yet unpublished) method for quantifying fluorophore with a wide field, but not a confocal, microscope. You would need a pure sample of the fluorophore (in your case, may be PFA-fixed GFP) for calibration. But if fixation reduces its fluorescence to a variable degree it will not work of course. Anyway, if that’s of interest to you, please contact me directly.

 

Mike

 

Michael Model, Ph.D.

Confocal Microscopy,

Dpt Biological Sciences,

1275 University Esplanade,

Kent State University, Kent, OH 44242

tel. [phone]

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Young Jik Kwon
Sent: Wednesday, April 22, 2009 11:20 PM
To: [hidden email]
Subject: Re: GFP tissue preparation for confocal microscopy

 

Hi Cam,

Thanks for the information. We are trying to quantify gene expression by fluorescence intensity. Do you think PFA would work for such work?

Best,


Young

 

On Wed, Apr 22, 2009 at 8:01 PM, Cameron Nowell <[hidden email]> wrote:

Hi Young,

 

PFA fixation should not totally kill your GFP, it may weaken it a bit but not totally wipe it out. If you find you have lost all your fluorescence after fixation you can always go in with an anti-GFP antibody tagged with Alexa488 or similar and get the signal back.

 

I have fixed numerous tissues and cells in 4% PFA and seen very little degradation of the signal. For more aggressive staining (like BrdU incorporation that requires acid fixation) the signal is gone, this is when the anti-GFP antibody is useful.

 

 

Cheers

 

Cam

 

 

Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: [phone]
Mobile: [phone]
Fax: [phone]

Facility Website

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Young Jik Kwon
Sent: Thursday, 23 April 2009 12:34 PM
To: [hidden email]
Subject: GFP tissue preparation for confocal microscopy

 

All,

We are trying to take confocal micrographs of at tumor tissues that express GFP. What would be the sample preparation procedures? We tried cryosectioned samples but the cell morphology seems weird. We already know paraformaldehyde fixation kills GFP fluorescence. Any expert's advice?

Best,

Young

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