Differential extraction of proteins from paraformaldehyde-fixed cells: lessons from synaptophysin and other membrane...

While investigating the localization of synaptophysin in PC12 cells using immunofluorescence microscopy, we noticed a striking difference in its apparent subcellular distribution depending on whether digitonin or Triton X-100 was used as permeabilization agent of paraformaldehyde (PFA)-fixed cells. We found that this difference was due to epitope inaccessibility in the digitonin-treated cells combined with an almost quantitative extraction of the antigen on Triton X-100 permeabilization. Both phenomena were differential with respect to the various synaptophysin-containing compartments. The extraction of antigen from PFA-fixed cells was also seen with other membrane proteins but not with cytosolic proteins and proteins in the lumen of the secretory pathway. Significantly, some of the membrane proteins were extracted from the PFA-fixed cells in higher-molecular-weight forms which we believe represent their in vivo oligomeric states. The implications of our observations are discussed with respect to the method of immunofluorescence microscopy and also to the possible use of paraformaldehyde as an in vivo crosslinker for the study of membrane protein quaternary structure.