安徽医科大学生命科学学院 School Of Life Sciences Aukui Wedieal Hniversity 细胞生物学实验之 微丝染色及形态观察 By生物学系-李超 2016-04 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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[目的要求] 1.掌握微丝的染色方法。 2.了解光镜下微丝的基本形态结构。 3.了解考马斯亮蓝染料在细胞生物技术中的 应用。 2 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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[实验原理] 1.细胞骨架 254m 25*m 25m INTERMEDIATE FILAMENTS MICROTUBULES ACTIN FILAMENTS 中间纤维 微管 微丝 3 鱼 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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[实验原理] 2.微丝 肌动蛋白，actin 负端 G-actin 三维结构 细胞松弛素B CH2 H3Cx.. H H CH3 与0nm 正端 HN F-actin F-actin 电镜照片 分子模型 粉 4 座 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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[实验原理] 3.染色 应力纤维 TritonX-100 考马斯亮蓝 处理 R250染色 ●溶解质膜和细胞内 ● 非特异性地将蛋白 许多蛋白质 染色 细胞骨架中蛋白质 ● 胞质背景着色弱 不被破坏 5 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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[实验用品] 材料：盖玻片培养的成纤维细胞 器具： 观察：倒置显微镜、普通光学显微镜 培养：CO2恒温培养箱、盖玻片、小平皿 操作：吸管、镊子、培养皿、吸水纸、烧杯、载玻片 试剂： 培养及细胞处理：6mmol/LPBS(pH6.5)、 1%TritonX-100 溶液、M-缓冲液(pH7.2)、DMEM培养液、多聚赖氨酸。 固定：3%戊二醛固定液 染色：0.2%考马斯亮蓝R250染液 6 渔 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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[实验步骤] 1.培养在成纤维细胞进行 传代培养时，将已消毒的盖 玻片涂上多聚赖氨酸，放入 培养皿中，于37℃、5%C02 的温箱中生长24h~48h。 PBS缓冲液 洗去培养液 2.取材 取出铺有细胞的盖 玻片，放入小培养皿中（正 面朝上)，用PBS洗涤3次， 洗去表面的培养液。 1%TritonX-100 抽提细胞膜系统及 非细胞骨架蛋白 3.抽提 吸弃PBS,加入1% Triton X-100溶液4~5滴( 刚好覆盖盖玻片表面)，置 M缓冲液 于室温10min。 稳定细胞骨架 4.稳定 吸弃1%Triton X 100，立即用M-缓冲液轻轻 地洗涤3次。 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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[实验步骤] 5.固定 吸弃M-缓冲液 ，略晾干，在3%戊二醛液 3%戊二醛 中固定10min,再以PBS 固定整个细胞 洗涤3次，洗去固定液。 6.染色 滴加3-5滴0.2% 考马斯亮蓝 考马斯亮蓝染液染色 R250染色液 应力纤维染色 15min,然后小心地用自 来水漂洗。 7.镜检在载玻片中加 滴自来水，将盖玻片有材 料一面朝下倒扣封片，吸 水纸吸去多余水分。制成 显微观察 临时装片，光镜下观察。 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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[注意事项] 1.洗片时要轻柔，以免把细胞从盖玻片上洗去。 2.操作中应注意区分细胞盖玻片的正反面。 3.染色后应冲洗盖玻片背面，避免损伤细胞。 4.TritonX--100抽提蛋白时，注意避免抽提时间过 长而导致细胞结构的破坏。 9 This document is produced by trial version of Print2Flash.Visit www.print2flash.com for more information
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