Fixation of Embryonic Mouse Tissue for Cytoneme Analysis.
Fixation of Embryonic Mouse Tissue for Cytoneme Analysis.
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DOI：
10.3791/64100
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发表时间：
2022-06-16
期刊：
影响因子：
--
通讯作者：
Ogden SK
中科院分区：
文献类型：
--
作者：
Hall ET;Daly CA;Zhang Y;Dillard ME;Ogden SK
发育组织模式和发育后组织稳态取决于称为形态发生素的细胞信号的受控传递。形态发生素以浓度和时间依赖性方式发挥作用，指定指导和加强细胞命运的不同转录程序。确保适当的形态发生素信号阈值的一种机制是通过称为细胞线的特殊丝状伪足传递信号蛋白。细胞体非常薄（直径≤200 nm），可以长到几百微米的长度，这使得它们的保存用于固定图像分析具有挑战性。本文描述了一种精细处理小鼠胚胎的方法，用于固定、免疫染色和厚切片，以便使用标准共聚焦显微镜观察细胞线。该协议已成功用于可视化在小鼠神经管发育过程中连接不同细胞信号传导室的细胞因子。该技术还可以适用于检测跨组织类型的细胞因子，以促进以前所未有的分辨率询问发育信号。这里，提供了一个优化的分步方案，用于胚胎的固定、免疫染色和切片，以检测发育中的小鼠组织中称为细胞线的特殊信号丝状伪足。
Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (≤200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution. Here, an optimized step-by-step protocol is provided for fixation, immunostaining, and sectioning of embryos to detect specialized signaling filopodia called cytonemes in developing mouse tissues.
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