Re: problem with fresh frozen eGFP sample

Posted by Haberman, Ann on
URL: http://confocal-microscopy-list.275.s1.nabble.com/problem-with-fresh-frozen-eGFP-sample-tp593098p593099.html

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Dear Jean,

We struggled with this exact issue for quite a while before we found the solution on the internet (embedded in someone's blog about their frustrations!).

My understanding is that acetone or 2-methybutane achieve fixation by dehydrating tissue. As a result, that fixation process extracts cytoplasmic GFP. For that reason, any stains for water soluble proteins are best performed with PFA or formalin fixed tissue. That is the case for many proteins found within the cytoplasm.

However, GFP does not fluoresce well after formaldehyde fixation so many investigators have included an additional anti-GFP stain to detect the presence of GFP that no longer can fluoresce! Rockland makes an anti-GFP that also detects YFP.

best,

Ann

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Hi all,

I have an animal that is Tg eGFP for a neurotransmitter in astrocytes.  It shows up very well in brain and spinal cord after perfusing with 4%PFA.  I'm trying to Laser capture some cells for RNA work but having trouble visualizing the cells in the fresh frozen tissue.  I'm doing cervical dislocation, the disection is under 1min., then placing the tissue into 2-Methylbuane that has been cooled and surrounded by dry ice.  The tissue is then cut in a cryostat, 8um section placed onto a slide.  I then visualize using either laser or mercury bulb (with 488 filter) and the cells look abstract or green everywhere instead of dark background with green astrocytes (star shaped cells).  I have cut frozen sections from PFA perfused animals several times and there is no problem with the way the cells look.  Do you think I need to hydrate the slide to visualize the cells better?  Is the freezing method faulty?  Any suggestions?

Thanks for your help.
Jean


Jean Brennan
Research Specialist
Rothstein lab, Dept of Neurology
Johns Hopkins University
600 N. Wolfe St., Meyer 6-174
Baltimore, MD  21287
410-614-4119; FAX: [phone]
[hidden email]

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Hi all,

 

I have an animal that is Tg eGFP for a neurotransmitter in astrocytes.  It shows up very well in brain and spinal cord after perfusing with 4%PFA.  I'm trying to Laser capture some cells for RNA work but having trouble visualizing the cells in the fresh frozen tissue.  I'm doing cervical dislocation, the disection is under 1min., then placing the tissue into 2-Methylbuane that has been cooled and surrounded by dry ice.  The tissue is then cut in a cryostat, 8um section placed onto a slide.  I then visualize using either laser or mercury bulb (with 488 filter) and the cells look abstract or green everywhere instead of dark background with green astrocytes (star shaped cells).  I have cut frozen sections from PFA perfused animals several times and there is no problem with the way the cells look.  Do you think I need to hydrate the slide to visualize the cells better?  Is the freezing method faulty?  Any suggestions?

 

Thanks for your help.

Jean

 

 

Jean Brennan
Research Specialist
Rothstein lab, Dept of Neurology
Johns Hopkins University
600 N. Wolfe St., Meyer 6-174
Baltimore, MD  21287
410-614-4119; FAX: [phone]
[hidden email]

-- 

Ann Haberman, PhD
Department of Laboratory Medicine
Yale University School of Medicine
1 Gilbert  St.
TAC S541
New Haven, CT 06510

203-785-7349
203-785-5415 (fax)
[hidden email]