Cryosectioning and immunolabeling
Cryosectioning and immunolabeling
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DOI：
10.1038/nprot.2007.365
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Geuze, Hans J.
中科院分区：
文献类型：
--
作者：
Slot, Jan W.;Geuze, Hans J.
在本协议中，我们描述了蛋白质和某些脂质的亚细胞定位的冷冻免疫标记方法。该方法首先在甲醛（FA）和/或戊二醛（GA）中对细胞和组织进行化学固定，有时还添加丙烯醛。细胞和组织块浸泡在2.3 M蔗糖中，然后在液氮中冷冻。薄的冷冻切片，在超核组中切割，可以用不同大小的金颗粒进行单个或多个免疫标记，对比并在电子显微镜下观察。半薄冷冻切片可用于免疫荧光显微镜。我们详细描述了过去几十年来在我们实验室开发和实践中测试的程序。
In this protocol, we describe cryoimmunolabeling methods for the subcellular localization of proteins and certain lipids. The methods start with chemical fixation of cells and tissue in formaldehyde (FA) and/or glutaraldehyde (GA), sometimes supplemented with acrolein. Cell and tissue blocks are then immersed in 2.3 M sucrose before freezing in liquid nitrogen. Thin cryosections, cut in an ultracryotome, can be single- or multiple immunolabeled with differently sized gold particles, contrasted and viewed in an electron microscope. Semi-thin cryosections can be used for immunofluorescence microscopy. We describe the detailed procedures that have been developed and tested in practice in our laboratory during the past decades.