I. LOSS OF LIPIDS DURING PREPARATION O AMOEBAE FOR ELECTRON MICROSCOPY
I. LOSS OF LIPIDS DURING PREPARATION O AMOEBAE FOR ELECTRON MICROSCOPY
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DOI：
10.1016/0005-2760(66)90013-0
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发表时间：
1966-01-01
期刊：
影响因子：
--
通讯作者：
WEISMAN, RA
中科院分区：
文献类型：
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作者：
KORN, ED;WEISMAN, RA
将变形虫单独或组合固定在戊二醛、高锰酸钾或四氧化锇中，并用乙醇溶液、环氧丙烷和包埋溶液提取，为电子显微镜检查做准备。对每种溶剂提取的脂质进行化学测量（酯和磷分析），并使用在[H3]-棕榈酸存在下生长的变形虫进行放射性分析。通过硅酸色谱法将提取的脂质分级为中性脂质和磷脂，并测定各自的脂肪酸组成。在戊二醛中固定后，脂质没有变化，并且在脱水过程中几乎完全被乙醇提取。高锰酸盐固定后，用固定剂和乙醇提取所有中性脂质和约2±5％的磷脂。在四氧化锇中固定后，用乙醇和包埋介质提取大部分中性脂质和一些磷脂。四氧化锇破坏了不饱和脂肪酸。结论是，如果采用常规的固定和脱水方法，则不可能在电子显微镜水平上进行脂质代谢的放射自显影研究。此外，单独固定在戊二醛中后在细胞中可视化的结构在观察时可能不包含脂质。
Amoebaewere fixed in glutaraldehyde, potassium permanganate or osmium tetroxide, alone or in combination, and extracted with ethanolic solutions, propylene oxide and embedding solution as in preparation for electron microscopy. The lip id that was extracted by each solvent was measured chemically (ester and phosphorus analyses) and by radioactive analysis using amoebae grown in the presence of [H3] -palmitic acid. The extracted lipids were fractionated by silicic acid chromatography into neutral lipids and phospholipids and fatty acid composition of each was determined. After fixation in glutaraldehyde, the lipids were unchanged and were almost completely extracted by ethanol during the dehydration procedure. After permanganate fixation, all of the neutral lipid and about 2 5% of the phospholipid were extracted by the fixative and ethanol. Following fixation in osmium tetroxide, most of the neutral lipid and some of the phospholipid were extracted by ethanol and the embedding medium. Osmium tetroxide destroyed the unsaturated fatty acids. It is concluded that autoradiographic studies of lipid metabolism at the electron microscopic level are not possible if the usual methods of fixation and dehydration are employed. Also, structures visualized in the cell after fixation in glutaraldehyde alone probably do not contain lipid at the time of observation.