凍結置換法

freeze substitution

[目次：試料等(試料および試料作製)]

急速凍結固定法(加圧凍結法、金属圧着凍結法)により固定した生物試料内の非晶質氷をアセトン等の有機溶媒で置換し、脱水を行う方法。試料の微細構造の破壊を防ぐため、置換は数日かけて-80 ℃から4 ℃まで段階的に温度を上昇させて行う。その際、有機溶媒に四酸化オスミウムや酢酸ウラン等を加えることで、化学固定および電子染色を同時に行う場合が多い。置換後は試料を室温まで戻し、樹脂包埋後、超薄切片を作製し、電顕観察を行う。

Freeze substitution is a technique to replace amorphous ice with an organic solvent (acetone, etc.) (dehydration) in a biological specimen fixed by rapid freeze fixation, where the specimen is subject to rapid freeze fixation (high pressure freezing or metal mirror freezing (slam freezing)).  To prevent destruction of fine structures of the specimen, the substitution is carried out by raising temperature step-by-step from -80 ℃ to 4 °C over a few days.  In the course of the substitution, chemical fixation and electron staining are often performed by adding osmium tetroxide and/or uranium acetate.  Then, the substituted specimen is returned to room temperature and is subject to resin embedding.  A TEM specimen is made by ultrathin sectioning the specimen.

関連用語から探す