SUBCELLULAR-LOCALIZATION OF SV2 AND OTHER SECRETORY VESICLE COMPONENTS IN PC12 CELLS BY AN EFFICIENT METHOD OF PREEMBEDDING EM IMMUNOCYTOCHEMISTRY FOR CELL-CULTURES

We demonstrated the subcellular localization of SV2, a transmembrane p rotein associated with neuroendocrine secretory vesicles, in NGF-treat ed PC12 cells by preembedding EM immunocytochemistry (ICC), using a sm all gold probe followed by silver enhancement. The use of a multiwell chamber slide substantially improved the efficiency of the preembeddin g EM ICC procedures for cell cultures. The advantages and related cave ats of this method ate discussed. SV2 was distinctly localized on dust ers of synaptic vesicles and large dense-cored vesicles (LDCV). The di stribution of SV2 on these two types of secretory vesicles was compare d quantitatively to that of another secretory vesicle-associated trans membrane protein, synaptophysin. In cultures under similar experimenta l conditions, the ratio of SV2 vs synaptophysin ICC staining on synapt ic vesicle clusters was about 1:1, whereas it was about 9:1 on LDCV me mbranes. Furthermore, whereas SV2 is localized on the membranes of the LDCVs, chromogranin A, an acidic protein in secretory granules, is de arly in the core of the LDCVs. This is the first demonstration of thes e two antigens in such dose (similar to 20 nm) yet distinct compartmen ts within a single organelle.