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Live cell staining should be done on ice if possible to prevent endocytosis of the antibody during the staining. Fixation should follow staining and then if necessary the application of a secondary antibody.
Best regards,
Sandy
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Sandra K. Masur, PhD
Professor, Ophthalmology
Mount Sinai School of Medicine, Box 1183
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On Aug 21, 2012, at 3:55 PM, Julia Edgar wrote:
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I agree. Live cell staining (without fixation or permeabilisation) circumvents this problem.
Julia
Yes, you do get partial permeabilisation with para formaldehyde.
You can stain unfixed non permeabilised cells to look at membrane proteins.
Michelle
On 21 Aug 2012, at 20:38, "Trop, Stefanie A." <[log in to unmask]> wrote:
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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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