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| 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 |
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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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