Whole Mount Preparation of Primary Cultured Neuron for HVEM Observation

High-voltage electron microscope (HVEM) has higher resolution and penetration power than conventional transmission electron microscope that could be load thick specimen. Some researchers have taken this advantage of HVEM to explore 3-dimensional configuration of the biological structures including tissue and cells. Whole mount preparations has been employed to study some cell lines and primary culture cells. In this study, we would like to introduce useful whole mount preparation method for neuronal studies. The plastic coverslips were punched, covered by formvar membrane and coated with carbon. The neurons obtained embryonic 18 rat hippocampus were seeded on the prepared cover slip. The coverslips were fixed, dried in freeze drier and kept in a descicator until HVEM observation. We could observe detailed neuronal structures such as soma, dendrite and spine under HVEM without conventional thin section and heavy metal stain. The anaglyphic image based on stereo paired image ($-8^{\circ},+8^{\circ}$) provides three dimensional perception of the neuronal dendrites and their spines. This method could be applied to sophisticated analysis of dendritic spine under the various experimental conditions.

배양된 시경세포 관찰을 위한 초고압전자현미경 홀마운트 시료제작기법