Correlative Microscopy for Localization of Proteins In Situ: Pre-embedding Immuno-Electron Microscopy Using FluoroNanogold Gold Enhancement and Low- Temperature Resin

摘要

Immuno-electron microscopy (immuno-EM) is a technique that has been used widely to determine sub-cellular localization of proteins. Different approaches are available for immuno-EM: pre-embedding method, post-embedding, and cryosectioning (Tokuyasu “style”). Here we describe a pre-embedding technique that allows the labeling of a target protein in situ, retention of fluorescence signal in plastic, and its localization at the EM level in a given cellular context. The procedure can be technically challenging and labor intensive: it requires optimization of fixation protocols to better preserve the cellular morphology and screening of compatible antibodies. Nevertheless, immuno-EM can be a powerful localization tool.