Fig. 1. SNAREs are clustered in plasma membrane sheets derived from PC12 (A–H) or BHK cells (I and J). (A and B) Fixed sheets were immunostained for syntaxin 1 (A) followed by staining with FM1-43 (B) to visualize phospholipid membranes. Syntaxin is distributed in numerous brightly fluorescent dots (A). Fluorescence arising from stained syntaxin can not be seen through the brighter FM1-43 fluorescence (B). (C and D) Unfixed membrane sheets were stained for syntaxin 1 and imaged (C). They were then fixed and re-stained (D). Closed circles indicate syntaxin clusters on fixed membrane sheets that were already positive on the unfixed membrane sheet; dashed circles indicate syntaxin clusters that were visible only after fixation. Note that often a higher concentration of dots was observed at the rim of the membrane sheets (C and D), which is probably due to some membrane curling at the edges. (E and F) Imaging of an unfixed membrane sheet derived from a PC12 cell expressing syntaxin 1A–GFP (E), followed by immunostaining for syntaxin 1 (F). Arrows highlight spots that remained unchanged after antibody labeling. (G and H) Fixed membrane sheets double-stained for syntaxin 1 (G) and SNAP-25 (H). Spots positive for syntaxin 1 are positive (closed circles) or negative (dashed circles) for SNAP-25. (I and J) Unfixed membrane sheets immunostained for syntaxin 4 (I) followed by FM1-43 staining (J). Fluorescent spots on membranes derived from BHK cells were less numerous than syntaxin 1-positive spots on PC12-cell membranes.