Transmission electron microscopy fixation

Microscopical methods light microscopy - transmission electron microscopy (TEM) - scanning electron microscopy (SEM) - perfusion fixation - transmission electron microscopy (TEM) - scanning electron microscopy (SEM)... 

III. Transmission electron microscopy of radish seeds Transmission electron microscopy (TEM) of radish seeds was done as listed below For TEM preparations, the specimens after fixation and dehydration, were embedded in Epon 812 resin (Luft, 1961). Thick sections (ca. 1mm each) were stained with 0.1% toluidine blue and observed with a Zeiss light photomicroscope. Thin sections, obtained with a diamond knife on a Supernova microtome, were sequentially stained at room temperature with 2% uranyle acetate (aqueous) for 5 min and by lead citrate for 10 min (Reynolds, 1963). Ultrastructural studies were made using a Philips CM12 transmission electrone microscope (TEM) operated at 80 KV. 

Chemical fixation for transmission electron microscopy prepares cells for the preservation of damage due to subsequent washing with aqueous solvents, dehydration with organic solvents such as ethanol or acetone, embedding in plastic resins, polymerization of the resins by heat, exothermic catalysts, or ultraviolet radiation, and imaging with high-energy electron beams in an electron microscope. 

List of Fixation Conditions for Preparation of Plant Cells and Tissues for Transmission Electron Microscopy... 

FIGURE 1.1. Electron micrographs of liver tissue processed differently for transmission electron microscopy. (A) Processed by the conventional method. Note superior quality of ultrastructural preservation compared with that obtained with microwave heating. (B) Rapidly processed by vacuum microwave heating. The whole process from tissue fixation to resin embedding was completed in 2 hr. The quality of ultrastructural preservation is satisfactory. Magnification 6,21 Ox (B). (B) courtesy of Richard T. Giberson. 

Transmission, freeze-fracture, and scanning electron microscopy have contributed ultrastmctural information about passive transport, particularly within the stratum comeum. Advances in fixation protocols for transmission electron microscopy have preserved the intercellular bilayers of the SC and have corrobrated their role in both passive and enhanced dmg delivery. Scanning and freeze-fracture electron microscopy have supplemented transmission electron microscopy by supplying three-dimensional representations of the transport pathways within the SC. 

Fig. 8.9. Human platelets after the radiolabeling procedure, examined by transmission electron microscopy. Almost no signs of platelet activation, manifested as pseudopodia formation. Perfusion fixation, critical point drying, x 40 500...

Transmission electron microscopy (TEM) is important to monitor cell-free sperm decondensation and nuclear formation. To prepare specimens for TEM, incubation mixture aliquots were fixed for 45 min in 2.5% (v/v) paraformaldehyde, 3.1% (v/v) glutaraldehyde, 0.02% (w/v) picric acid in 30 mAf NaHP04, pH 7.5. After fixation, samples were embedded in 2% (w/v) low-gelling-temperature agarose and postfixed for 15 min in 1% (w/v) OSO4. Samples may be dehydrated either in ethanol and propylene oxide or acetone and embedded for sectioning in Spurr s low-viscosity epoxy resin (Spurr, 1%9). Before examination, sections of about 70-nm thickness should be stained, e.g., with uranyl acetate and Reynolds lead citrate (Re)molds, 1%3). 

Figure 3. Morphology by transmission electron microscopy of the VLDPE of Figs. 1 and 2, after staining/fixation by chlorosulphonation. The inset shows a 1.6 times magnified view of a more severely treated sample revealing the internal morphology of the CSDs.

This protocol is used for light microscopic analysis of the adult eye (Tomlinson and Ready 1987), and it provides nice preservation of pigment granules and photoreceptor rhabdomeres therefore, it is primarily used for phenotypic and mosaic analysis of adult eyes (Figure 13.2). Because this fixation method does not preserve either cell membranes or subcellular structures, transmission electron microscopy (TEM) is recommended for visualizing these structures. 

Ultrastructural Analysis. Samples to be examined by electron microscopy were prepared as described by Persi and Burnham (19). All samples were fixed using the following schedule glutaral-dehyde (4% (w/v), 6h), osmium tetroxlde (20% (w/v), 6h) tannic acid (10% (w/v), 3h), and osmium tetroxlde (20% (w/v), 2h). Each fixative was prepared In 0.2M cacodylate buffer, pH 7.2, and samples were washed between fixations with cacodylate buffer. Subsequently, all cells were treated with uranyl acetate, washed, dehydrated through a graded ethanol series, and finally embedded In Maraglas (Polysciences, Inc.). Ultrathln sections were cut, post-stained with lead citrate and examined in a Phillips 300 transmission electron microscope operating at 60 Kv. 

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