Immunogold Labeling of Cryosections from High‐Pressure Frozen Cells
Immunogold Labeling of Cryosections from High‐Pressure Frozen Cells
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DOI：
10.1111/j.1600-0854.2007.00552.x
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发表时间：
2007-05
期刊：
影响因子：
4.5
通讯作者：
E. V. van Donselaar;G. Posthuma;D. Zeuschner;B. Humbel;J. Slot
中科院分区：
文献类型：
--
作者：
E. V. van Donselaar;G. Posthuma;D. Zeuschner;B. Humbel;J. Slot
根据Tokuyasu(Tokuyasu KT.一种对细胞悬液和组织进行超速切割的技术。J Cell Biol 1973；57：551-565)，是一种重要且广泛使用的免疫电子显微镜方法。这些切片是从在室温下化学固定(室温固定，RTF)的材料切割而成的。最近，在许多形态学研究中，用快速冷冻和冷冻置换固定(CSF)取代了RTF。我们探索了一些新的方法将免疫金标记法应用于高压冷冻细胞(HepG2细胞、原代软骨细胞)和组织(软骨和外分泌胰腺)的冰冻切片。由于免疫标记必须在解冻和稳定的切片上进行，我们探索了两种方法来实现这一点：(1)切片固定法，如前面简要报道的(Liou W et al.Organochem Cell Biol 1996；106：41-58和Möbius W et al.《组织化学细胞化学杂志》2002；50：43-55。将新鲜冷冻细胞的冰冻切片稳定在蔗糖和甲基纤维素以及不同浓度的戊二醛、甲醛和醋酸铀酰(UA)的混合物中。只有偶尔，这种方法才会显示细胞保存良好的切片区域和负染色的膜，比如RTF材料的Tokuyasu切片。(Liou等人)(2)再水化方法是一种新的方法，即用戊二醛和/或四氧化三铯(OsO4)处理脑脊液，然后进行再水化和冷冻，就像在Tokuyasu方法中那样。特别是在脑脊液中添加尿酸和低浓度的水有利于获得极佳的膜对比度。免疫金标记法与Tokuyasu法一样有效。
Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. A technique for ultracyotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–565), is an important and widely used method for immunoelectron microscopy. These sections are cut from material that is chemically fixed at room temperature (room temparature fixation, RTF). Lately in many morphological studies fast freezing followed by cryosubstitution fixation (CSF) is used instead of RTF. We have explored some new methods for applying immunogold labeling on cryosections from high‐pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). As immunolabeling has to be carried out on thawed and stable sections, we explored two ways to achieve this: (1) The section fixation method, as briefly reported before (Liou W et al. Histochem Cell Biol 1996;106:41–58 and Möbius W et al. J Histochem Cytochem 2002;50:43–55.) in which cryosections from freshly frozen cells were stabilized in mixtures of sucrose and methyl cellulose and varying concentrations of glutaraldehyde, formaldehyde and uranyl acetate (UA). Only occasionally does this method reveal section areas with excellent cell preservation and negatively stained membranes like Tokuyasu sections of RTF material. (Liou et al.) (2) The rehydration method, a novel approach, in which CSF with glutaraldehyde and/or osmium tetroxide (OsO4) was followed by rehydration and cryosectioning as in the Tokuyasu method. Especially, the addition of UA and low concentrations of water to the CSF medium favored superb membrane contrast. Immunogold labeling was as efficient as with the Tokuyasu method.