Download Electron Microscopy: Methods and Protocols by John J. Bozzola (auth.), John Kuo (eds.) PDF
By John J. Bozzola (auth.), John Kuo (eds.)
The revised and extended moment variation of this publication provides the most recent know-how in electron microscopy, whereas keeping the practicality and accessibility of the acclaimed first variation. just like the first version, this quantity offers transparent, concise directions on processing organic specimens and comprises dialogue at the underlying ideas of the vast majority of the procedures provided. Electron Microscopy contains significant components of electron microscopy-transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The TEM sector covers a number of key strategies, together with: traditional specimen education tools for aesthetic cells and biomedical and plant tissues; cryospecimen practise via high-pressure freezing and cryoultramicrotomy unfavorable staining and immunogold labeling thoughts; and TEM crystallography and cryo-TEM tomography. The SEM region equally attends to conventional-, variable pressure-, environmental-, and cryoscanning microscopy options, in addition to the appliance of X-ray microanalysis. Protocols for the applying of X-ray microanalysis to SEM and mass spectrometry finish the volume.
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Extra info for Electron Microscopy: Methods and Protocols
7. PO traditionally is used as a transitional solvent for facilitating infiltration following dehydration and prior to embedding (1,5). PO is the best organic solvent for decreasing the viscous character of the epoxy resins used for formulating embedding media. It is necessary to reduce the viscosity of the embedding media to increase infiltration capability into the tissue. PO, however, can severely deplete lipids from fixed tissues and is a known carcinogen. The loss of lipids, and perhaps other cell components as well, is exacerbated by lengthy, stepwise infiltration schedules that gradually reduce the concentration of ethanol while increasing that of PO.
4. Notes 1. Cell cultures, isolated cells, pellets, plant materials, or biopsy specimens easily can be fixed in situ or by simple immersion (5). Organ systems that are easily accessible, such as liver or heart, can be removed rapidly and also processed by immersion (3). However, this choice is not the best in this instance because fixation by immersion depends on diffusion of the fixative into the tissue, a rather slow process. TBVP, although it is lengthy and technically more difficult, is the method of choice for preparing animal organ tissues (16).
The excision method may be chosen by individuals who lack experience with TBVP or who are not working with tissues taken from the central nervous system. Similar reagents as were used in TBVP are also used in the excision-immersion method but in a different manner. 26 Mascorro and Bozzola 1. The animal may be anesthetized or humanely sacrificed. Tissues are surgically removed as rapidly as possible to prevent artifact formation, or degradation of ultrastructural detail. Generally, a portion of the tissue is removed and placed into a container of fixative, usually a Petri dish with buffered GA such as a 2% solution of GA in Sorenson’s buffer.