Methods and principles of fixation by freeze-substitution.
Methods and principles of fixation by freeze-substitution.
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DOI：
10.1083/jcb.4.5.593
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发表时间：
1958-09-25
期刊：
影响因子：
--
通讯作者：
SIDMAN RL
中科院分区：
文献类型：
--
作者：
FEDER N;SIDMAN RL
冷冻置换是基于组织的快速冷冻，然后在远低于0 °C的温度下用冰溶解（“置换”）。将1至3 mm的试样投入到由液氮冷却至-175 ° C的3：1丙烷-异戊烷中。(with预防措施）。将冷冻组织置于-70 ° C的替代液中。1周，使冰缓慢溶解，而不会使组织结构变形。冲洗掉过量的替代剂，并将标本包埋、切片和常规染色。为了获得最佳的形态学和组织化学保存，替代液通常应含有化学固定剂和冰溶剂，例如，1%的四氧化锇丙酮溶液、氯化汞乙醇溶液和苦味酸乙醇溶液。在单独的溶剂中取代后，结构的保持较差。已获得的证据表明，化学剂在低温下可固定组织。固定的化学机制可能类似于在室温下操作的化学机制：含有固定剂的新化学交联将组织成分连接在一起。这一过程不同于纯溶剂的变性。冷冻置换法有许多优点，特别是在光学显微镜的分辨率极限下保持结构，以及许多可溶性和不稳定物质的准确定位。
Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O°C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175°C. (with precautions). The frozen tissue was placed in substituting fluid at -70°C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances.
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影响因子：
--
作者：
CASON, JE
通讯作者：
CASON, JE
DOI：
10.1083/jcb.4.4.459
发表时间：
1958-07-25
期刊：
The Journal of biophysical and biochemical cytology
影响因子：
--
作者：
LADMAN AJ
通讯作者：
LADMAN AJ
DOI：
10.1016/s0074-7696(08)60004-2
发表时间：
1952-01-01
影响因子：
--
作者：
BELL, LGE
通讯作者：
BELL, LGE
影响因子：
--
作者：
ADAMSTONE, FB;TAYLOR, AB
通讯作者：
TAYLOR, AB
DOI：
10.1098/rspb.1955.0067
发表时间：
1955-01-01
期刊：
PROCEEDINGS OF THE ROYAL SOCIETY SERIES B-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BARTER, R;DANIELLI, JF;DAVIES, HG
通讯作者：
DAVIES, HG