Smith Alyson S, Nowak Roberta B, Fowler Velia M
Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA.
Methods Mol Biol. 2018;1698:205-228. doi: 10.1007/978-1-4939-7428-3_12.
During erythropoiesis, erythroblasts undergo dramatic morphological changes to produce mature erythrocytes. Many unanswered questions regarding the molecular mechanisms behind these changes can be addressed with high-resolution fluorescence imaging. Immunofluoresence staining enables localization of specific molecules, organelles, and membrane components in intact cells at different phases of erythropoiesis. Confocal laser scanning microscopy can provide high-resolution, three-dimensional images of stained structures, which can be used to dissect the molecular mechanisms driving erythropoiesis. The sample preparation, staining procedure, imaging parameters, and image analysis methods used directly affect the quality of the confocal images and the amount and accuracy of information that they can provide. Here, we describe methods to dissect erythropoietic tissues from mice, to perform immunofluorescence staining and confocal imaging of various molecules, organelles and structures of interest in erythroblasts, and to present and quantitatively analyze the data obtained in these fluorescence images.
在红细胞生成过程中，成红细胞经历显著的形态变化以产生成熟红细胞。利用高分辨率荧光成像可以解决许多关于这些变化背后分子机制的未解答问题。免疫荧光染色能够在红细胞生成不同阶段的完整细胞中定位特定分子、细胞器和膜成分。共聚焦激光扫描显微镜可以提供染色结构的高分辨率三维图像，可用于剖析驱动红细胞生成的分子机制。所使用的样品制备、染色程序、成像参数和图像分析方法直接影响共聚焦图像的质量以及它们所能提供的信息量和准确性。在这里，我们描述了从小鼠身上分离红细胞生成组织的方法，对成红细胞中各种感兴趣的分子、细胞器和结构进行免疫荧光染色和共聚焦成像的方法，以及呈现和定量分析这些荧光图像中获得的数据的方法。
