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Localization of specific proteins within cells at the nanometer level of resolution is central to understanding how these proteins function in cell processes such as motility and intracellular trafficking. Such localization can be achieved by combining transmission electron microscopy (TEM) with immunogold labeling. Here we describe a pre-embedding, indirect gold immunolabeling approach to localize two different proteins of interest with secondary antibodies labeled with gold particles of different sizes in cells grown on cover slips. In this protocol, the cells are immunolabeled prior to being embedded in an epoxy resin for ultrathin sectioning. The protocol also includes strategies for optimizing the balance between ultrastructure and antigen preservation, steps to minimize nonspecific antibody binding, and steps to optimize antibody penetration.
Since Faulk and Taylor  took advantage of the high electron density of gold to map the distribution of Salmonella antigens by transmission electron microscopy (TEM) and Frens  developed methods to control the size of colloidal gold particles, several thousand studies have used variations of these basic protocols to visualize, at the ultrastructural level, the localization of specific proteins in cells and tissues. Combined with the results of molecular, biochemical, and fluorescence light microscopy approaches, the results of these ultrastructural studies have contributed to our understanding of the function of numerous proteins. In fact, immunogold electron microscopy is the only morphological method with sufficient spatial resolution to provide definitive localization of proteins within organelles such as lysosomes, mitochondria, or synapses (e.g., [3–5]) or the association of protein linkers with organelles or cytoskeletal polymers (e.g., [5–9]).
While immunogold TEM on tissues enables investigators to determine protein localization in situ, transfection of cultured cells enables them to localize protein domains or tagged proteins and to use correlative microscopy to determine the distribution of specific proteins in the same cell at the light and electron microscope levels [10, 11]. Another advantage of using cultured cell monolayers is that they are often thinner than tissue sections, thereby facilitating antibody penetration. The protocol presented in this chapter considers only cultured cell monolayers since 3D cultures, depending on their thickness, may be more similar to tissue sections and should be processed as such for immunogold labeling for TEM. Cultures of non-adherent, floating cells can also be processed for immunogold TEM using protocols developed for tissue sections after collecting these non-adherent cells as a pellet by centrifugation 
Immunolabeling can be direct or indirect. In direct immunolabeling, the primary antibody is conjugated to gold particles. In indirect immunolabeling, the primary antibody is unconjugated and labeling is achieved with a gold-conjugated secondary antibody specific for the primary antibody. The advantage of the indirect procedure is that multiple secondary antibodies can bind to the primary antibody increasing the signal. The disadvantage is that the gold particles are further away (15–30 nm) from the site the primary antibody binds to on the antigen.
Immunolabeling can also be performed pre- or post-embedding. In post-embedding protocols, the cells are fixed with a strong cross-linking chemical, usually glutaraldehyde, then embedded in epoxy or low-viscosity resin and sectioned with an ultramicrotome. The sections obtained are then immunolabeled. In pre-embedding protocols, the cells are fixed with a weak fixative, usually formaldehyde, followed by immunolabeling. The cells are then embedded in epoxy resin and sectioned with an ultramicrotome. Post-embedding protocols using cells embedded in epoxy resin offer excellent ultrastructural preservation but often result in poor labeling because protein epitopes are altered by the glutaraldehyde fixation and the high temperature associated with the curing of the epoxy resin during embedding. This results in reduced primary antibody binding and thus a lower signal. Pre-embedding protocols offer better epitope preservation, better antibody binding, and thus a stronger signal. The trade-off of the pre-embedding protocol is reduced ultrastructural preservation because the fixatives used are weaker than those used in post-embedding protocols.
The pre-embedding protocol described in this chapter utilizes a post-labelling fixation step to minimize ultrastructural loss. In this step, immunolabeled cells are subsequently fixed with glutaraldehyde and osmium in order to stabilize the antibody complexes and to prevent ultrastructural degradation that occurs during the dehydration and heating steps associated with embedding in epoxy resins. This methodology for indirect double immunolabeling allows the distribution and localization of two proteins to be determined simultaneously. Combined with biochemical and genetic analysis, double immunolabeling provides another method to demonstrate the interaction of two proteins and to locate the cellular regions or organelles where this interaction occurs [6, 9].
Drs. Skalli, Estraño, and Schwartzbach are supported by in-house grants from the University of Memphis. J.C.R. Jones research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number RO1 AR054184; the content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.
1A large selection of established cell lines is commercially available through vendors such as the American Type Culture Collection. Countless protocols have been published describing how to establish primary cell cultures from organs. Prior to carrying out cell culture procedures, secure necessary authorizations regarding biosafety and/or use of animal or human tissues to derive primary cell cultures. The methods presented in this protocol for human endothelial cells can be used with modification of culture media for virtually any cell type. Consult the literature or vendor’s instructions to determine which one is best suited for your cell culture system.
2The advantage of Thermanox plastic cover slips over glass cover slips is that they can be peeled off the epoxy during the embedding procedure whereas glass cover slips need to be shattered with liquid nitrogen or dissolved with hydrofluoric acid, procedures which may damage the cells embedded in the epoxy. However, if using glass cover slips, ensure that they are not too thick (#1 or #1.5 is fine); otherwise they may not shatter easily with liquid nitrogen during the embedding procedure.
3All solutions should be prepared with distilled water and stored at 4 °C unless otherwise stated.
4Paraformaldehyde, glutaraldehyde, and osmium tetroxide are toxic and volatile. All work with these chemicals should be performed in a fume hood using gloves and protective clothing. Handling and waste disposal should be done according to the guidelines of the local authorities.
5Acetylated BSA or BSA-c (Electron Microscopy Science, Hatfield, PA) at a final concentration of 0.2% can be used in lieu of BSA in the dilution buffer for the primary and secondary antibodies and may be more efficient than BSA at reducing nonspecific immunogold staining.
6Preparation of cells for immunolabeling denatures cellular proteins. The antibodies used must thus be able to recognize epitopes on denatured proteins. Only use a primary antibody whose specificity and ability to recognize denatured proteins with high specificity have been verified by Western blotting and fluorescent immunolabeling of cells fixed with 4% paraformaldehyde.
7Direct labeling using primary antibodies conjugated with different size gold particles can be used in place of the indirect labeling procedure (i.e., using gold-conjugated secondary antibodies for detection), outlined here. One advantage in doing so is that gold particles attached to the primary antibody are closer to the site of antigen than in the indirect procedure where gold particles are separated from the antigen by both primary and secondary antibody molecules. Nonetheless, our preference is the indirect protocol, since multiple secondary antibodies binding to a single primary antibody result in amplification of the gold label signal output.
8This reagent is not needed if performing direct labeling.
9The 2 % OsO4 solution can be stored at 4 °C for several months provided that it is kept in the dark. Discard the solution if a black precipitate forms. OsO4 fumes are harmful and all procedures involving OsO4 should be done in a fume hood and OsO4 wastes should be disposed of in closed containers.
10To enhance membrane visualization, a 1.5% aqueous solution of potassium ferricyanide (K3Fe(CN)6) can be added to the 1 % OsO4 fixation solution . The mixing of these two solutions should be performed immediately prior to use and the fixation should be undertaken at 4 °C for 4 h in the dark.
11UA is toxic and radioactive. Due caution should be taken with its handling. Long-term exposure, skin contact, and inhalation should be avoided.
12Embedding medium based on Epon 812 was routinely used until the 1970s. However, in 1978 Epon 812 was discontinued. Nonetheless, a number of companies subsequently developed Epon 812 equivalents which are now used in embedding medium formulations. We indicate one such example.
13Unused epoxy resin embedding mix can be stored for up to 6 months at −20 °C but it is preferable to use a freshly prepared mix. Epoxy resin waste should be collected and allowed to polymerize before disposal.
14TEM grids are available in different mesh sizes. The smaller the mesh size, the better the sections will be supported by the grids and able to resist tearing when exposed to the electron beam. However, too small a mesh size can result in parts of the section being obscured. With larger mesh sizes, such as the commonly used 200, the sections are more likely to tear under the electron beam. To help preventing this shortcoming, grids coated with a formvar/carbon film can be used. However the formvar/carbon film will sometimes reduce image contrast. Therefore, decision on what mesh size to use and whether or not to use grids coated with a formvar/carbon film is mostly a matter of personal preference.
15Carry out the procedures described in this section by using sterile techniques and working in a laminar flow biosafety cabinet.
16To determine cell concentration with a hemocytomer, slowly pipet 20 μL of cell suspension under the edge of the cover slip sitting above the hemocytometer gridded area. Then, place the hemocytometer on the stage of a light microscope and count the cells in the central gridded square. The cell concentration (cells/mL) is calculated with the formula (total cells counted × dilution factor × l04). Cell concentrations can also be determined with automated cell counters, but they are more expensive than hemocytometers.
17The purpose of this step is to create a “handle” which can be used to carry the cover slip from one solution to another during the immunogold staining. The tweezers need to be sterile for this procedure and this is achieved either by autoclaving or by, dipping the tip of the tweezers in 95 % ethanol and flaming it
18The cover slip may float due to air bubbles forming at the bottom of the well when the dish is placed in the incubator. If this happens, remove the dish from the incubator and with sterile tweezers gently push the cover slip down toward the bottom of the dish to dislodge the bubbles.
19If the cells are too sparse, it will be difficult to find them when performing TEM observations.
20Procedures in this section are carried out at room temperature and do not need to be performed under sterile conditions unless otherwise noted.
21Cell fixation that may be optimal for immunofluorescence, such as fixation/extraction in −20 °C acetone or a mix of acetone and methanol, does not preserve high-quality ultrastructure. However, antigenicity may not be preserved in cells fixed with standard TEM fixatives such as 2.5% glutaraldehyde. Thus, compromises must be made between preserving antigen-binding sites while maximizing the preservation of cellular architecture structure. Compromises include fixation in 4% paraformaldehyde or 4% paraformaldehyde plus 0.1–0.2% glutaraldehyde. Immunofluorescence can be used to test whether or not the aldehyde fixation solution preserves the antigenicity of the protein being labeled.
22Alternatively, the specimen can be incubated in 0.5 mg/mL sodium borohydride (NaBH4) in PBS for 20 min to reduce the free aldehyde groups that can nonspecifically bind colloidal gold.
23Permeabilization is not needed for extracellular epitopes. Increasing permeabilization time and/or detergent concentration will result in degradation of cellular membrane and membranous organelle ultrastructure. Some protocols employ 0.1 % NP40 in PBS in lieu of Triton-X-100.
24A 24-well plate is used at this stage to reduce cost by minimizing the amount of antibody used.
25It is useful to start with the dilution of primary antibody that gives good consistent staining in sections prepared for immunofluorescence microscopy. To reduce nonspecific binding of the secondary antibody to the specimen, serum of the animal species in which the secondary antibody is made should be added to the primary antibody diluent at a dilution of 1:20–1:100. To determine the extent of nonspecific immunogold labeling, experiments should be performed by omitting the primary antibody. Additional controls may also involve using a primary antibody known not to recognize proteins of the species from which the specimen was taken.
26This step is not needed if performing direct labeling (see Note 7).
27PBS and TBS are optimal for antibody binding, but may form precipitates with uranyl acetate, osmium, and/or lead citrate and thus must be replaced by EM wash buffer at this stage. EM wash buffer is not used to dilute antibodies in previous steps because it is not optimal for antibody binding to antigen. An alternative to EM wash buffer is 0.1 M phosphate buffer, pH 7.2.
28Propylene oxide will dissolve most plastic dishes. The plastic of Thermanox cover slips, however, is not dissolved by propylene oxide.
29If the cells were grown on glass cover slips, the glass cover slips can be removed (shattered) from the BEEM capsule by immersion in liquid nitrogen or dissolved with hydrofluoric acid.
30Alternatively, slice the sides of the BEEM capsule with a razor blade to loosen the block but this carries a. higher risk to damage the block.
31The fixed cells may also be visible as a black or brown line using a binocular dissecting microscope. This line can also be used for orientation.
3250–70 nm Ultra-thin sections will appear to have a silvery shine when floating on the surface of the water and viewed with the binocular mounted on the ultramicrotome.
33Sections can be dried and viewed with the transmission electron microscope prior to staining with heavy metal salts (uranyl acetate and lead citrate) to determine whether the immunolabeling was efficient. Gold particles, especially small ones (5 nm), may be more readily visualized in the absence of heavy metal staining and in that case heavy metal staining may be omitted. However, biological structures may be difficult to identify in the absence of heavy metal staining.
34Uranyl acetate staining enhances the electron contrast of biological structures by interacting with lipids and proteins. However, needle-like crystal precipitates may be present in uranyl acetate solutions that have not been cleared by filtration or centrifugation or may form in the presence of residual PBS on the section.
35Lead citrate staining enhances the electron contrast of biological structures by interacting with proteins and glycogens. Lead citrate, however, forms precipitates of lead carbonate in the presence of CO2. The sodium hydroxide pellets adsorb the CO2 preventing precipitate formation.