By Gérard Ailhaud
Adipose tissue is now famous as a widely-dispersed secretory organ that performs an important function in weight problems. Ailhaud (U. de Nice-Sophia Antipolis, great, France) and a crew of eu and US laboratory specialists and clinicians element the type, key recommendations hired, and their capability difficulties, within the in vivo and in vitro examine of brown and white fats tissues and cells from the dietary, physiological, and molecular degrees. significant therapy is accorded to the isolation, subcellular fractionation, and gene move into mature adipocytes, in addition to metabolic elements. Illustrations contain microscopy learn effects, and a guy donning a conveyable detector for measuring adipose tissue blood circulate.
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Depending on fixation and final storage, magnification may be satisfactory in the 10–15 K (K = ×1000) range; the zoom of the morphometric apparatus is useful when cristae are very closely packed, for instance, when multilocular cells are strongly activated (13). By a macrosequence specifically programmed for this purpose, the procedure is started by tracing mitochondrial profiles on the screen (to obtain their surfaces in µm2). Then, after tracing the cristae from one side to the other, the software adds up the various length measurements (in µm) and calculates their density (in µm/µm2).
Tech. 26, 526–527. 37. Willison, J. H. M. and Rowe, A. T. (1980) Replica, shadowing and freeze-etching techniques, in Practical Methods in Electron Microscopy, vol. 8. (Glauert, A. ), North-Holland, Amsterdam, The Netherlands. 38. , and Björntorp P. (1985) Human brown adipose cells in culture. Exp. Cell Res. 159, 261–266. 39. , and Zancanaro, C. (1986) Ultrastructure of brown adipocytes mitochondria in cell culture from explants. J. Submicrosc. Cytol. 18, 625–627. 40. , and Bosello, O. (1983) Ultrastructural features of cultured mature adipocytes precursors from adipose tissue in multiple symmetric lipomatosis.
3. Methods The methods to obtain single cell suspensions of white and brown adipose cells from the rat ATs are described in detail in Chapters 5, 10, 15, and 24, and in refs. 11 and 12. Cells are incubated with insulin or other compounds at 37°C. Isolated rat adipose cells in suspension (floating cells) are used throughout the following staining protocols. ICC can be performed using direct or indirect techniques. Direct methods use a labeled specific (primary) Ab to bind directly (in one step) to the cellular epitope.