Re: GFP tissue preparation for confocal microscopy

Posted by Cameron Nowell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-tissue-preparation-for-confocal-microscopy-tp2680696p2680906.html

Hi Young,

 

Quantifying fluorescence intensity is always difficult, if not impossible. You will be able to measure relative shifts in intensity between samples providing they are all treated the same, stained the same and captured using the same settings (eg. Exposure time or gain setting etc.).

 

PFA should be fine for all that. My recommendation is to purchase PFA that is supplied in ampoules. We use a 16% PFA that is stored in an ampoule under nitrogen gas. This is because PFA will oxidise to methanol fairly quickly. This could be why you are seeing GFP loss with your fixation, your fixative maybe all methanol by now.

 

 

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 1:20 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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This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.