By Gérard Ailhaud
Adipose tissue is now famous as a widely-dispersed secretory organ that performs an important position in weight problems. Ailhaud (U. de Nice-Sophia Antipolis, great, France) and a crew of ecu and US laboratory specialists and clinicians aspect the type, key suggestions hired, and their power 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 remedy is accorded to the isolation, subcellular fractionation, and gene move into mature adipocytes, in addition to metabolic features. Illustrations contain microscopy learn effects, and a guy donning a conveyable detector for measuring adipose tissue blood circulate.
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Additional resources for Adipose Tissue Protocols
Introduction The isolated rat adipose cell experimental system is the principal model for studies of the mechanism of insulin’s stimulatory action on glucose transport (reviewed in refs. 1–4). Despite the successful use of this preparation in biochemical studies, the unique structure of the adipose cell, with its large central triglyceride storage droplet (80 µm) and thin (1–2 µm) rim of cytoplasm, has caused special problems for morphological approaches. Several methods have been used for studies of protein localization and trafficking by immunocytochemistry (ICC) at both light microscopy (LM) and electron microscopy (EM) levels.
2. BROWN ADIPOCYTE COUNT Brown adipocytes can be counted exclusively at the morphologic level, particularly when tissues are mixed. Because of the presence of several small lipid vesicles, recognizing cell profiles is difficult. One possible solution is to count only cells in which the nucleus is visible: brown adipocyte nuclei are easily distinguished from the others by their round shape, and are often located at the center of the cell. To demonstrate BAT recruitment resulting from cold acclimation in old rats (44), we have correlated absolute numerical values, adopting the following procedure: 1.
At this stage, the immunologic step of the reaction can be performed: sections are incubated with the Ab that recognizes as Ag the DIG molecule present in the previously formed hybrids. This Ab is directly conjugated to the alkaline phosphatase enzyme, which reacts with NBT/BCIP, producing a purple-blue precipitate. Sections are incubated with the Ab for 2 h at RT, Morphologic Techniques for BAT and WAT 37 washed to remove excess Ab and incubated in the dark in a solution containing NBT/BCIP. The colorimetric reaction requires monitoring and incubation from a minimum of 2 h to a maximum of 3 d, depending on the amount of transcript.
Adipose Tissue Protocols by Gérard Ailhaud