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 staff of eu and US laboratory specialists and clinicians aspect the category, key concepts hired, and their capability difficulties, within the in vivo and in vitro learn 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 points. Illustrations comprise microscopy research effects, and a guy donning a transportable detector for measuring adipose tissue blood move.
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4, for 1–2 h at RT and frozen in freon cooled with liquid nitrogen. Specimens are fractured, and replicated at –112°C in a Balzers freeze-fracturing device (Balzers, Lichenstein). Tissue is then digested with Na hypochlorite, and with a chloroform–methanol mixture and 100% dimethyl formamide, which are used specially for ATs. Replicas are washed with dH2O and recovered on 200-mesh grids (37). 8. 1. ) Fixation and postfixation are performed for TEM and SEM with the same fixatives and procedures as adopted for tissues, except that these operations last only about 30 min.
J. Cell Sci. 103, 931–942. 14. Hayat, M. , ed. (1974). Biological application, in Principles and Techniques of Scanning Electron Microscopy, vol. 2. Van Nostrand Reinhold, London. 15. , and Cinti, S. (1994) Quantitative evaluations of gap junctions in old rat brown adipose tissue after cold acclimation: a freeze-fracture and ultrastructural study. Tiss. Cell 26, 667–676. 16. , and Bosello, O. (1985) Ultrastructural study of adipocyte precursors from epididymal fat pads of adult rats in culture.
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. Indirect methods comprise at least two steps: first an unlabeled primary Ab binds to the specific cellular epitope; in a second step the bound primary Ab is detected by a labeled-secondary Ab.