Detection of Cell Wall-Anchoring Machinery by Immunogold-Labeling Thin-Section Electron Microscopy

Cell wall anchoring of surface proteins and pili in Gram-positive bacteria is mediated by sortase - a highly conserved transpeptidase enzyme. Early studies have demonstrated the membrane-associated nature of this enzyme in close proximity with its cognate substrates, using immunogold-labeling thin-section electron microscopy. Here, we provide a detail protocol of this methodology, including specimen preparation, ultrathin sectioning, and immunogold-labeling electron microscopic procedures, with an experimental model of sortase enzymes from Actinomyces oris. In principle, this protocol can be employed for any bacterial ultrathin-section samples to detect subcellular localization of proteins and organelles by immuno-electron microscopy.