Visualization of the Nuclear Lamina in Mouse Anterior Pituitary Cells and Immunocytochemical Detection of Lamin A/C by Quick-freeze Freeze-substitution Electron Microscopy
Visualization of the Nuclear Lamina in Mouse Anterior Pituitary Cells and Immunocytochemical Detection of Lamin A/C by Quick-freeze Freeze-substitution Electron Microscopy
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DOI：
10.1369/jhc.4a6478.2005
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发表时间：
2005-04
影响因子：
3.2
通讯作者：
T. Senda;A. Iizuka-Kogo;Atsushi Shimomura
中科院分区：
文献类型：
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作者：
T. Senda;A. Iizuka-Kogo;Atsushi Shimomura
采用电镜技术结合冷冻置换、深度刻蚀和免疫细胞化学对快速冷冻的垂体前叶细胞的核层进行了观察，并与化学固定的细胞进行了比较。通过速冻冷冻取代电子显微镜，在核膜内部发现了一层厚达20nm的电子透光层，而在传统的戊二醛固定细胞中则看不到这层。通过速冻深蚀刻电镜，我们无法明确区分新鲜未固定或戊二醛固定细胞的核层对应层。免疫荧光显微镜显示，在丙酮固定细胞和多聚甲醛固定细胞中，细胞核中检测到纤层蛋白A/C，而在长时间多聚甲醛固定细胞中，细胞核中未检测到纤层蛋白A/C。在快速冷冻和冷冻替代的细胞中，免疫金电镜也显示了层状蛋白A/C的核定位，而在多聚甲醛固定的细胞中则没有。Lamin A/C主要分布于距离核膜60 nm内的外周核质中，与核膜相对应。这些结果表明，与传统的化学固定相比，速冻固定可以在超微结构和免疫细胞化学上保存核层。
We examined the nuclear lamina in the quickly frozen anterior pituitary cells by electron microscopic techniques combined with freeze substitution, deep etching, and immunocytochemistry and compared it with that in the chemically fixed cells. By quick-freeze freeze-substitution electron microscopy, an electron-lucent layer, as thick as 20 nm, was revealed just inside the inner nuclear membrane, whereas in the conventionally glutaraldehyde-fixed cells the layer was not seen. By quick-freeze deep-etch electron microscopy, we could not distinguish definitively the layer corresponding to the nuclear lamina in either fresh unfixed or glutaraldehyde-fixed cells. Immunofluorescence microscopy showed that lamin A/C in the nucleus was detected in the acetone-fixed cells and briefly in paraformaldehyde-fixed cells but not in the cells with prolonged paraformaldehyde fixation. Nuclear localization of lamin A/C was revealed by immunogold electron microscopy also in the quickly frozen and freeze-substituted cells, but not in the paraformaldehyde-fixed cells. Lamin A/C was localized mainly in the peripheral nucleoplasm within 60 nm from the inner nuclear membrane, which corresponded to the nuclear lamina. These results suggest that the nuclear lamina can be preserved both ultrastructurally and immunocytochemically by quick-freezing fixation, rather than by conventional chemical fixation.