High-Resolution Imaging of Cellular Proteins

Jonathan C. R. Jones

Steven D. Schwartzbach, Omar Skalli, Thomas Schikorski

Despite the improved resolution capacities of fluorescence microscopy over the last 20 years, localization of specific proteins at the ultrastructural level with gold-conjugated antibodies remains a valuable technique in the cell biological tool chest. Ultrastructural immunolocalization of specific proteins in tissues rather than in cultured cells is often advantageous because, in tissues, the interactions between different cell types and with the extracellular matrix are maintained. Here, we describe two immunogold labeling procedures to localize at the ultrastructural level one or more proteins. In the first procedure (pre-embedding), micrometer-thick tissue cryostat sections are immunostained prior to embedding for obtaining ultrathin sections suitable for TEM, while in the second procedure (post-embedding), tissues are embedded in a hydrophobic resin such as Lowicryl K4M and ultrathin sections are first obtained and then immunolabeled. While the former method is better at generating strong immunolabeling, the latter is better at preserving ultrastructure.