Deep-etching immunogold replica electron microscopy of cytoskeletal elements in cultured hamster heart cells

A procedure has been developed for the three-dimensional immunoelectron microscopic localization of cytoskeletal filaments by a deep-etching replica method in combination with immunogold labeling and/or myosin subfragment 1 (S1) decoration techniques. Neonatal hamster heart cells grown on glass coverslips were extracted with Triton X-100 or physically permeabilized by breaking open the cell membranes. S1 decoration was performed on some specimens immediately after the permeabilization. After prefixation in formaldehyde, samples were immunostained with poly- or monoclonal antibodies to desmin or vimentin, and indirectly tagged with colloidal gold probes by the biotin-streptavidin method. After postfixation with glutaraldehyde, tannic acid and osmium tetroxide, the cells were freeze-etched and rotary-replicated with platinum and carbon in a freeze-fracture apparatus. Replicas were viewed with a transmission electron microscope using a tilting specimen stage to obtain stereo images. The procedure made it possible to identify the specific filaments within the complex cytoskeletal networks in cultured hamster heart muscle and nonmuscle cells at high resolution and in three dimensions. The method has advantages in its three-dimensionality and feasibility to evaluate the data by comparing them with those obtained by alternative light microscopic methods. Details of the protocol and a description of the results of using three different antibodies are given.