By Gerard Aihaud
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Extra resources for Adipose Tissue Protocols (Methods in Molecular Biology Vol 155)
4, 4 h at 4°C. 2. Wash overnight in PB. 3. 4, 1 h at 4°C. 4. 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.
J. Ann. Diabetol. Hôtel Dieu 125–129. 36. Weigle, D. S. (1997) Leptin and other secretory products of adipocytes modulate multiple physiological functions. Ann. Endocrinol. 58, 132–136. 37. , Pinkney, J. , and Coppack, S. W. (1998) Adipose tissue as an endocrine and paracrine organ. Int. J. Obesity 22, 1145–1158. 38. , and Ricquier, D. (1997) Leptin gene is expressed in rat brown adipose tissue at birth. FASEB J. 11, 382–387. 39. , Zingaretti, M. , and Cinti, S. (1998) Leptin and UCP1 genes are reciprocally regulated in brown adipose tissue.
1. Sample Collection Sample collection for TEM studies is critical, because of the artifacts that may be introduced in this phase. This is primarily because samples for fixation must be very small (approx 1 mm3), and are therefore difficult to manipulate. 5 cm3, and then reduced with a razorblade to fragments not exceeding 1 mm3. When collecting a tissue sample, particular care must be exercised in identifying with precision the sampling area. The macroscopic features of the zone of collection must be kept in mind, because, unlike LM analysis, TEM examination is performed on tissue fragments that are much too small to allow subsequent orientation, given the inherent absence of microscopic topographic references.