文章摘要
不同固定方法处理小鼠视网膜行免疫荧光染色的效果比较
Comparison of Immunofluorescence staining Results on the Retina of Mice treated with Different Fixation Methods
投稿时间:2018-09-23  修订日期:2018-10-14
DOI:
中文关键词: 小鼠  视网膜  灌注固定  浸泡固定  免疫荧光组织化学染色
英文关键词: mice  The retina  Perfusion fixation  Immersion fixation  Immunofluorescence histochemical staining
基金项目:陕西省自然科学基金2018KW-063
作者单位E-mail
刘子赫 空军军医大学(第四军医大学) A15904538329@163.com 
贾宁 空军军医大学(第四军医大学)延安大学医学院  
贾瑞华 空军军医大学(第四军医大学)西京医院  
李瑞 空军军医大学(第四军医大学)基础医学院  
王璐 空军军医大学(第四军医大学)延安大学医学院  
高方 空军军医大学(第四军医大学)基础医学院 gao_fang_2011@163.com 
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中文摘要:
      目的:免疫荧光组织化学染色(immunofluorescence histochemistry, IFHC)在神经生物学研究中具有十分广泛的应用,包括在视网膜中的应用。为了获得最优的IFHC结果,我们对神经视网膜的固定方法进行优化。方法:以小鼠为观察对象,对比灌注法(Perfusion, P)、浸泡固定方法(Immersion, I)以及两者结合(I+P)的方法,分为以下几组:灌注组(P),浸泡固定2小时组(I2),浸泡固定12小时组(I12),浸泡固定24小时组(I24),灌注+浸泡后固定2小时组(P+I2),灌注+浸泡后固定12小时组(P+I12)。获取不同固定方法处理后的视网膜组织,蔗糖脱水后进行冰冻切片,观察切片的整体形态并进行IFHC染色(包括抗Pax6抗体染色、抗rhodopsin抗体染色),再以荧光二抗标记,激光共聚焦扫描获得图片。对比各组最终所获图片,分析染色效果。结果:P+I2组和P+I12组的效果最好,视网膜组织结构最完整,IFHC方法行Pax6和rhodopsin的染色效果良好。结论:针对小鼠的神经视网膜进行IFHC实验时,灌注结合后固定12小时,可以作为标准的最优的组织固定方法。
英文摘要:
      Objective: Immunofluorescence histochemistry (IFHC) is widely used in neurobiological studies, including in the retina. In order to obtain optimal IFHC results, we optimized the fixation method of the neural retina. Methods: mice as the observation object, contrast infusion method (Perfusion, P), soak the fixed methods (based, I) and the (I + P) of the combination of methods, divided into the following groups: r (P), soak a fixed group (I2) 2 hours, soak the fixed set of 12 hours (I12), 24 hours soak fixed group (I24), Perfusion + 2 hours after soaking fixed group (P + I2), Perfusion + 12 hours after soaking fixed group (P + I12). Retinal tissues treated with different fixation methods were obtained, frozen sections were performed after sucrose dehydration, the whole morphology of the slices were observed and stained with IFHC (including anti-pax6 antibody staining and anti-rhodopsin antibody staining), and the images were obtained by fluorescence secondary antibody labeling and laser confocal scanning. Compare the final images of each group and analyze the dyeing effect. Results: P+I2 group and P+I12 group showed the best results, with the most complete retinal tissue structure, and the IFHC staining results of Pax6 and rhodopsin were good. Conclusion: when IFHC experiment was performed on the neural retinas of mice, 12 hours of fixation after perfusion could be used as the standard optimal tissue fixation method.
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