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Gérard Ailhaud's Adipose Tissue Protocols PDF

By Gérard Ailhaud

Adipose tissue is now well-known as a widely-dispersed secretory organ that performs an important position in weight problems. Ailhaud (U. de Nice-Sophia Antipolis, great, France) and a workforce of ecu and US laboratory specialists and clinicians aspect the class, key strategies 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 elements. Illustrations comprise microscopy examine effects, and a guy donning a conveyable detector for measuring adipose tissue blood movement.

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3. Dehydrate in graded EtOH series: 75% 15 min, 95% 15 min, and 100% 2 × 15 min, stirring, at RT. Infiltrate with LR white (in the authors' experience with AT, the medium grade) at RT, 2–3 changes, 1 h each, then leave overnight. 4. Polymerization: the authors use thermal curing at 50°C for 24–48 h. It is important to limit the contact of oxygen with the resin during polymerization. The most convenient way of achieving this is to use gelatin capsules. 3. Tissue Sectioning Resin blocks are sectioned with an ultramicrotome, and ultrathin sections are placed on 300-mesh nickel grids.

Wash in 1X PBS. 2. METHOD 2: OSO4 MACERATION TECHNIQUE 1. Quick perfusion (when this is possible) with the fixative indicated at item 3. 2. Reduction of the tissue into thin strips (1 × 1 × 5 mm). 3. 1 M Na-cacodylate-HCl buffer for 15 min at RT in the dark. 4. 2. 5. 25% potassium ferrocyanide for 2 h (36). 6. Embedding in agarose and sectioning (about 150 µm) with a chopper microtome. 7. Washing in PBS. 8. 1% OsO4 in PBS, for 50 h at 20°C or for 3 h at 45°C. 9. Multiple rinses in PBS. Morphologic Techniques for BAT and WAT 41 10.

In our experience, positive controls, where available, are the more important, because they allow interpretation of the positive reaction and distinguish it from the background. One of the fundamental interpreting problems to be addressed when examining an IHC reaction is to understand whether the staining that is observed on a cell or a structure is produced by the Ag–Ab reaction, or is nonspecific (background). In case of uncertainty about the presence of internal controls in sections, it is advisable to add to the series of slides subjected to processing at least one slide containing a structure rich in the Ag that is being investigated.

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