A new method for electron microscopic observation of isolated synaptic vesicles labelled with monoclonal antibody

The immunoreaction of a monoclonal antibody (Mab) and an isolated synaptic vesicle (SV) was processed on a grid mesh and the result could be easily observed with electron microscopy. The SV suspension was obtained and dispersed on the grid mesh where immunoreaction procedures were performed. The resulting immunoreaction was visualized by labelling with ferritin particle (FAD) or horseradish peroxidase (HRP) for the electron microscopic observation. The SV specimen was observed by electron microscopy after faint negative staining with 1% uranyl acetate. With this method, the positive immunoreaction of Mab 171B5 and the isolated SV could be easily identified by the formation of a halo of FAD or a cobweb of HRP surrounding the SV. In the control experiment, the SV specimen was incubated with normal mouse serum instead of the Mab while the other procedures were performed in the same way. The SV was not outlined by FAD in the control experiment. Thus, the positive immunoreaction of the Mab and SV was thought to be an immunologically specific one. It was also determined that the Mab reacted specifically with the SV but not with the small membrane fragments and other unknown material. The present method seems to be useful for observing the immunoreaction of subcellular structures and their antibodies under electron microscopy.