C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining.
C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining.
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DOI：
10.3791/64204
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发表时间：
2022-09-16
期刊：
影响因子：
--
通讯作者：
Lee TW
中科院分区：
文献类型：
--
作者：
Ananthaswamy D;Croft JC;Woozencroft N;Lee TW
梭线虫生殖系是研究减数分裂的极好模型，部分原因是在解剖的动物上进行细胞学分析很容易。整个安装准备保存减数分裂细胞核的结构，重要的是，每个性腺臂包含减数分裂的所有阶段，组织在一个时空的进展，使它很容易识别在不同阶段的细胞核。成年雌雄同体有两个性腺臂，每个都组织成一个封闭的管，在远端封闭端有增殖的生殖系干细胞，在近端开放端有细胞化的卵母细胞，它们在子宫中心连接。解剖从体腔释放一个或两个性腺臂，允许整个减数分裂可见。在这里，提出了针对感兴趣的蛋白质的免疫荧光的通用方案，然后进行DAPI染色以标记所有染色体。年轻的成年人被固定在左旋咪唑和快速解剖使用两个注射器针头。在生殖细胞挤出后，样品在液氮中经历冷冻裂纹之前被固定，这有助于渗透角质层和其他组织。然后可以将样品在乙醇中脱水，再水化，并与一抗和二抗孵育。将DAPI添加到封固剂中的样品中，这使得DNA的可视化可靠，并且可以很容易地找到动物在荧光显微镜下成像。这种技术很容易被那些熟悉处理C的人采用。花了几个小时练习解剖方法后，该协议已被教授给在研究实验室工作的高中生和本科生，并被纳入一所文科大学的基于课程的本科生研究经验。
The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.
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影响因子：
1.9
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