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Yep, in my lab's experience you cannot have PFA fixation without permeabilization.  In some cases  an additional permeabilizing step improves staining, but some intracellular staining is inevitable after PFA.  

However, Ms. Trop's problem is the reverse.  I would not panic a priori if I saw ubiquitinated proteins on the plasma membrane, but as to why you might find EEA1 on the plasma membrane, you have a few options.  Your labmate may have overfixed her cells, made a poor batch of staining buffer or in some other way interfered with the antibody's specificity.  You may have a batch of antibody that is old, not very good or which targets the wrong species.  Finally, an event that causes a large amount of PI3P to appear on the plasma membrane, for example lots of simultaneous clathrin-mediated endocytosis, could cause EEA1 to localize there temporarily.  In fact the many things that might cause EEA1 to go to the PM would probably involve an increase in ubiquitinated proteins on the PM as well.  

To sort these possibilities out, try starting with a western blot.  If you get a clean band from this cell line at about 150-160 kDa, re-make the IF buffer solutions and try again.  Include cells with different treatment conditions (e.g., serum-starved for a while) to rule out the possibility that this is a weird positive result.  

cheers, 


TF

Timothy Feinstein, PhD
Postdoctoral Fellow 
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine 
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Aug 21, 2012, at 4:45 PM, Christian Soeller wrote:

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> Some years ago we tried staining an intracellular protein without permeabilization, just two % pfa for 10 minutes. We got beautiful staining, indistinguishable from that in explicitly permeabilized cells. So antibodies can clearly get through and we have repeatedly verified that since then. Live cell surface labelling seems the way to go but requires an extracellular epitope.
> Christian
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> "MODEL, MICHAEL" <[log in to unmask]> wrote:
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> Formaldehyde normally makes membranes permeable only to some small molecules, not to antibodies. 
> 
> Mike Model
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Trop, Stefanie A.
> Sent: Tuesday, August 21, 2012 3:26 PM
> To: [log in to unmask]
> 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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