Procedure for Electron Microscopy

The sections are incubated for 72 hr at 4°C with primary antiserum in PBS containing 2% normal serum and 0.25% gum arabic (Sigma). The sections are then incubated overnight at 4°C with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), diluted 1 200. They are next incubated overnight at4°C with avidin-biotinylated peroxidase complex (ABC) diluted 1 100 in PBS containing 0.25% gum arabic. Note that the peroxidase-antiperoxidase procedure can also be used. 

The bound peroxidase is visualized by reaction with a filtered solution of 0.05% DAB and 0.0005% hydrogen peroxide in PBS. After rinsing with 0.15 M sodium phosphate buffer (pH 7.4), the sections are postfixed with 0.25% osmium tetroxide in the same buffer for 1 hr and counterstained with 1% uranyl acetate in deionized water. The sections are dehydrated and flat-embedded in Epon according to standard procedures (Hayat, 2000a). Controls are processed as above, except that incubation in the absence of the primary antibody is carried out with PBS containing 2% normal serum and 0.015% Triton X-100. 

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Franke, W. W., Krien, S., and Brown, R. M., Jr., 1969, Simultaneous gluta-raldehyde-osmium tetroxide fixation with postosmication. An improved fixation procedure for electron microscopy of plant and animal cells, Histochemie, 19 162-164. 

Labeling antibodies with colloidal gold was developed for electron microscopy, but the procedure can be used for light microscopy as well. 

As mentioned, chemical fixation of plant cells has been reviewed many times (15-20) and the reader is referred to these citations for a variety of fixation procedures for preserving plant cells and tissues. One of the most recent references regarding the topic is that of Hopwood and Milne (21). Table 1 presents their recommendations regarding fixation of plant cells and tissues for electron microscopy. 

Since the work reported by McCartney et al. (9), ultrathin sections of other, more heterogeneous components and mixtures of components of coals of different rank have been prepared and observed. Procedures for minimizing artifacts have been learned and followed, and experience in observation has led to avoiding obvious faults. These sections were often not as large and continuous as those of homogeneous vitrinites, but adequate areas were available for electron microscopy. Observations of these various components revealed ultrafine structures of different size and form. Some of the structures can be correlated with those deduced from other direct or indirect study techniques others are unfamiliar and novel, and suggested interpretations are tentative. 

Labeling in the microwave oven is usually carried out at 37°C for 15 min. Longer durations and higher temperatures may result in undesirable changes in antibody concentration and molarity of the salts and pH. After heat treatment, the sections should be kept at room temperature for at least 2 min to stabilize the antibody-antigen complexes. The step-by-step procedure for microwave heat-assisted immunolabeling of resin-embedded thin sections for electron microscopy follows (Rangell and Keller, 2000) ... 

The sections are observed under a light microscope and representative DAB-labeled neurons, and neuronal processes in the region of interest are selected for study with the electron microscope. The sections are trimmed with a razor Wade into small pieces containing the immunoreactive neurons. Thin sections are cut for electron microscopy. The results of this procedure are shown in Figure 8.4. 

The miscibility of water and hquid carbon dioxide is very poor and an intermediate solvent has to be used to allow the replacement of water by carbon dioxide. In a procedure initially developed to prepare representative samples for electron microscopy, water is replaced by ethanol through exchanges with alcoholic solutions of increasing concentration. The alcogel prepared by a final exchange with absolute ethanol (Fig. 3c) is introduced in a pressure vessel in which liquid CO2 is admitted and replaces ethanol in the gel. The C02-impregnated gel is compressed and heated above the critical point of CO2 (31.05°C, 73.8 bar). Release of pressure above the critical temperature allows CO2 to be extracted without the formation of any liquid-vapor interface and a dried aerogel is formed (Fig. 3d). 

Bendayan M, Stephens H (1984) Double immunostaining procedures techniques and applications. In Polak JM, Varndell LM (eds) Immunolabelling for electron microscopy. Elsevier, New York, NY, pp 143-154... 

Procedures for Microscopic Observations. Ultrathin film specimens (ca. 700-1000 A thick were prepared for electron microscopy by placing a drop of 0.1% benzene or ethylbenzene solution on a microscope mesh coated with collodion and carbon and then evaporating the solvent as gradually as possible at 30°C. Ethylbenzene as a solvent was selectively good for Is but poor for EO segments whereas benzene was a good solvent for both segments. Similar studies were made recently by Kovacs et al. (15, 16), Kawai et al. (17), Skoulious et al. (18, 19, 20, 21), and Crystal et al. (22, 23). 

All steps of male pronuclear formation in vitro can also be visualized by electron microscopy. Procedures have been well established in the frog system (see, for example, Vigers and Lohka, 1991). In the surf clam, in v/fro-assembled nuclei have also been examined by electron microscopy as described in Longo et al. (1994). In the sea urchin, a method for preparing nuclei for electron microscopy has been reported (Collas and Poccia, 1995a). A recent procedure for preparing swollen male pronuclei is described below. 

Immediately, two problems are obvious in attempting to relate birefringence and microtubules. First, the birefringence must be preserved during preparation for electron microscopy. Spindle birefringence in some materials is said to be maintained by a modified fixation procedure (Inou and Sato, 1967), but the possible effect of later... 

Effect of Different Dehydrating Procedures on Retention of Radioactive Lipid in Adipose Tissue during Specimen Preparation for Electron Microscopy - ... 

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