Application of immuno- and affinity labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells

In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λ_em ∼550 nm) and far-red (λ_em ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 μm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.