Transmission electron microscopy of cultured cells: Optimized protocols for ultrastructural studies and...

Nanometer-scale localization of specific proteins within cells is essential for understanding their roles in diverse physiological and pathological cellular processes. Such high-resolution localization of protein epitopes can be achieved by combining transmission electron microscopy (TEM) with immunogold labeling. Several immuno-TEM approaches are available, including pre-embedding and post-embedding techniques, each offering distinct advantages and limitations when applied to cultured cells. One of the major challenges is preparation of cell culture samples embedded in resin in a manner that enables reliable ultrathin sectioning and efficient collection of sections. Here, we describe an optimized post-embedding immunogold labeling procedure for the localization of one or more proteins in both adherent and non-adherent cells cultured on plastic coverslips. This approach has been successfully applied in our previous studies and proved suitable for routine ultrastructural immunolabeling of cultured cells. Although the protocol is designed to achieve an optimal balance between the preservation of cellular ultrastructure and antigenicity, optimal results may still depend on the specific characteristics of the proteins under investigation and their localization within particular cellular compartments. Therefore, we also present a complementary procedure for the ultrastructural analysis of cultured cells that can support and extend immuno-TEM investigations.