Confocal Microscopy List - Re: GFP tissue preparation for confocal microscopy

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-tp2680696p2680758.html

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
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Royal Melbourne Hospital
Victoria, 3050
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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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