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I guess I was wrong. But what we have certainly checked is that permeabilization by PFA does not happen until the fixative is removed (Pelts et al, Biotechniques, 2011). Of course you add antibodies only after a wash... -
Mike
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From: Confocal Microscopy List [[log in to unmask]] on behalf of Paul Rigby [[log in to unmask]]
Sent: Tuesday, August 21, 2012 8:09 PM
To: [log in to unmask]
Subject: Re: Paraformaldehyde and permeabilization
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Hi All,
We get asked these questions all the time and I can say, without doubt, that paraformaldehyde fixation will permeabilise cell membranes. Whether paraformaldehyde fixation alone will give your antibodies access to your antigen epitope is a totally different question and permeabilisation may additionally be required. This is likely why coagulative fixatives (like methanol and acetone) work well in some situations since they likely cause both fixation and permeabilisation at the same time. Similarly, antigen retrieval (perhaps also able to be called protein tertiary structure disruption) may also be required to sterically allow antibody molecules access to the antigen epitope. Exactly what is required to get good antibody staining is multi-factorial and is definitely dependent upon the particular antibody antigen epitope combination.
Beware, there is a massive amount of misinformation (or missing information) in the literature about antibody staining. Read widely, do appropriate testing and use suitable controls before publishing.
Sorry, I'll get off my soapbox now.
Cheers all
Paul
Assoc. Prof. Paul Rigby
Centre for Microscopy, Characterisation & Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley WA 6007
Australia
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From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Trop, Stefanie A.
Sent: Wednesday, 22 August 2012 3:26 AM
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Subject: Paraformaldehyde and permeabilization
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Dear list,
Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!
Yours,
Stefanie Trop
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Stefanie Trop, PhD Candidate
Johns Hopkins Bloomberg School of Public Health Department of Molecular Microbiology and Immunology Laboratory of Jelena Levitskaya
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(410) 502-9134
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