Encyclopedia of Experiments: Biology
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-从显微镜幻灯片上的一滴缓冲液开始,将完整的蠕虫转移到液滴中。 一旦蠕虫到位,使用无绒毛组织浸泡并取出缓冲器。 通过用90%乙醇给样品洗澡来修复样品,使其在每次应用后蒸发。
当乙醇在最终冲洗后蒸发后,在样品中加入含有核酸染料DAPI的溶液。 DAPI优先与DNA小槽中的腺苷和胸腺素碱基结合。当绑定时,DAPI-DNA复合物吸收紫外线并发出可见的蓝光。
接下来添加防褪色防腐剂,延长样品的保质期。涂抹盖片并密封在幻灯片上。然后使用带有蓝色过滤器的荧光显微镜来可视化DAPI机体,根据细胞及其周期状态,DAPI可以代表单染色体、姐妹色度对或整个细胞核。在这个实验中,我们将计算Ocytes中的DAPI染色姐妹色度对,以识别卡诺哈布迪炎的四倍体菌株。
- 四倍体菌株有12对染色体,可以通过计算未受精卵母细胞中的DAPI染色体来验证。为此,将5 到 10 微升 M9缓冲器放入滑梯上,并将 6到 10个假定四倍体转移到液滴中。 在解剖显微镜下,小心地将 M9 的大部分浸入无绒清洁组织中。
然后将 10微升 90% 乙醇滴到蠕虫身上,看着乙醇完全蒸发。 蒸发完成后,重复添加乙醇并观察乙醇蒸发的过程。总共在四个应用中添加 10 微升。 最后一滴蒸发后,以每微升两毫微克或类似污渍添加六微升 DAPI。
对于幻灯片的长期存储,将蠕虫安装在市售或自制防褪色中。然后添加盖片,用指甲油密封边缘。指甲油干燥后,滑梯可以打分。 使用荧光显微镜和100倍放大倍数。
首先,找到最成熟的未受精卵母细胞, 紧邻精子,尚未进入精子或子宫。 这里的DAPI机构大概是单染色体对。 接下来,聚焦于卵母细胞的细胞核,并使用显微镜的精细对焦从上到下缓慢扫描,同时计算DAPI天体。 然后通过将焦点从下移到顶部来叙述同一核中的 DAPI 机构。
野生型卵母细胞平均有六个DAPI体。 12个DAPI身体的存在表明,这种菌株中的动物要么是局部的,要么是完整的四倍体。 分析每株至少10种动物。 通常染色体对会非常接近或接触,因此DAPI 体的数量通常小于染色体对的实际数量。
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