Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections.

Abstract

We describe two methods for staining resinless thin sections with antibodies and gold-conjugated second antibodies. Immunolocalization of specific proteins is a powerful tool for cell structure studies but current techniques do not develop its full potential. Immunofluorescence provides only low-resolution localization, whereas conventional thinsection electron-microscopy images and immunostains only the section surface. Resinless sections of extracted cell structures offer a simple and effective means of immuno-electron microscopy. Without embedding plastic or soluble proteins, the cell cytostructure produces high-contrast, three-dimensional images. Resinless sections of detergent-extracted cells are prepared by embedding in diethylene glycol distearate, sectioning, and removing diethylene glycol distearate before microscopy.In the first method of immunostaining, extracted cells were fixed and stained with antibodies before embedment, sectioning, removal of the embedding resin, and critical point drying.In the postembedment method, the sample was embedded and sectioned, the diethylene glycol distearate was removed, and the sample was rehydrated before antibody staining. With these techniques, specific proteins were localized with high resolution throughout the entire section. Stereoscopic micrographs of resinless sections revealed the precise localization of specific cytoskeleton and nuclear matrix proteins in three dimensions with unprecedented clarity.The localization of specific proteins with labeled antibodies is a fundamental technique for exploring cell structure. Cytoskeletal filaments were first displayed by immunofluorescent staining (1-4), profoundly changing our understanding of cell structure. Antibody-based localization achieves its highest resolution with the electron microscope. In the conventional technique samples are embedded in a resin that is cut into ultrathin sections. Antibodies stain the section surface wherever antigenic epitopes are exposed. An important advance in the conventional technology was the use of colloidal gold-conjugated second antibodies (5-7). This methodology is well suited for viewing cross sections of cell organelles and membrane systems. It is, however, severely limited for structures such as the cytoskeleton or nuclear matrix, whose form is apparent only when seen in three dimensions.The conventional thin section is inadequate for imaging the structural networks of the cell, showing only that portion of the sample appearing at the section surface. This is because the embedding plastic presents the same average electron scattering cross section as biological materials and thus masks cellular components contained in the section interior. Heavy metals stain the section surface forming the actual image. Filaments of the structural networks are seen only in cross section unless, as rarely happens, they are tangent to the section surface. However, there is a simple and effective alternative to the embedded section. Cell structures form high-contrast, three-dimensio...

DNA Damage-Dependent Nuclear Dynamics of the Mre11 Complex

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Localization of heterogeneous nuclear ribonucleoprotein in the interphase nuclear matrix core filaments and on perichromosomal filaments at mitosis.

“…Previous reports have described in detail the modified immunogold staining procedure for resinless sections (44). Conventional immunostaining was modified to retain the gold beads during the step of temporary embedding in DGD.…”

“…Caski cells were grown on a Mylar film and extracted sequentially to produce the core filaments. Further processing and embedment in DGD for resinless section was carried out on the Mylar film as described (44). The Mylar substrate is easily peeled from the DGD block, which is then cut in an ultramicrotome.…”

“…Core filaments, prepared from cultured cells, were labeled with the fAl2 monoclonal antibody and gold bead-conjugated second antibody (44). Ultrathin, embedment-free sections were prepared as described and viewed by transmission electron microscopy (44). Fig.…”

Binding of ATP to UAP56 is necessary for mRNA export

“…Control sections were not exposed to the first antibody. The second antibody was linked to 5-nm gold beads (Nickerson et al, 1990). Cells were then fixed with 2.5% glutaraldehyde (Ted Pella) in 0.1 M Sörensen's phosphate buffer, pH 7.3 (Glauert, 1991) at 4°C for 1 hour, then washed twice in the same buffer and held overnight at 4°C in the buffer.…”