| 刘子赫,贾 宁,贾瑞华,李 瑞,张正平,王 璐,张 坤,高 方.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[J].,2019,19(11):2066-2071 |
| 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：2018-10-28 修订日期：2018-11-23 |
| DOI：10.13241/j.cnki.pmb.2019.11.013 |
| 中文关键词: 小鼠 视网膜 固定方法 免疫荧光染色 Pax6 Rhodopsin |
| 英文关键词: Mice The retina Fixation Immunofluorescence staining Pax6 Rhodopsin |
| 基金项目:陕西省自然科学基金项目（2018KW-063） |
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| 中文摘要: |
| 摘要 目的：免疫荧光染色(ImmunofluorescenceStaining, IF)是进行视网膜研究的重要方法之一。为了获得最优的IF结果，我们对视网膜的固定方法进行优化。方法：以小鼠视网膜为观察对象，分别采用浸泡固定方法(Immersion, I)、灌注法(Perfusion, P)以及两者联合(I+P)的方法进行固定，以IF方法对Pax6和Rhodopsin等抗原进行染色，对比各组视网膜组织的IF染色效果。其中，具体分组包括：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)，灌注组(P)，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)。结果：从大体结构来看，P+I2组的固定良好率达100%，I12、I24、P+I12组的良好率达83.33%，I2组和P组较低，分别是16.67%和0%。从Pax6的IF染色来看，I12组的染色满意度达100%，P+I2组和P+I12组的满意度为83.33%，I2组、I24组和P组的满意度较低，为16.67%。从Rhodopsin的IF染色结果来看，P+I2组和P+I12组的染色满意度为83.33%，I24组为50%，I2组、I12组和P组都为0%。P+I12组的视网膜切片行NF-M、PKC-?琢和glutamine synthetase (GS)染色也可获得良好的阳性结果。结论：综合大体结构、Pax6染色以及Rhodopsin染色等结果，P+I2组和P+I12组的效果最好，视网膜组织结构完整，IF方法行Pax6和Rhodopsin等的染色效果良好。因此，针对小鼠的神经视网膜进行IF相关实验时，灌注结合浸泡后固定2至12小时，可以作为最优的组织固定方法。 |
| 英文摘要: |
| ABSTRACT Objective: Immunofluorescence staining (IF) is one of the fundamental methods to observe retina. In order to obtain optimal IF results, we optimized the fixation method for retina. Methods: Immersion fixation (I), perfusion fixation (P) and combination of both (I+P) were used to fix the mouse retina. IF staining was applied to stain the antigens of Pax6, Rhodopsin, etc. The results of all groups were compared. The groups included the following ones: one group with immersion for 2 hours (I2), one group with immersion for 12 hours (I12), one group with immersion for 24 hours (I24), one group treated with perfusion fixation (P), one group fixed both with perfusion and immersion for 2 hours (P+I2) and one group fixed both with perfusion and immersion for 12 hours (P+I12). Results: In terms of gross morphology, the rates of intact sections were 100% in P+I2 group, 83.33% in I12, I24 and P+I12 groups and as low as 16.67% and 0% separately in I2 and P groups. With regard to IF staining of Pax6, the rates of satisfactory staining were 100% in I12 group, 83.33% in P+I2 and P+I12groups and 16.67% in I2, I24 and P groups. About the IF staining of Rhodopsin, the rates of satisfactory staining were 83.33% in P+I2 and P+I12 groups, 50% in I24 group and 0% in I2, I12 and P groups. The IF staining of NF-M, PKC-α and glutamine synthetase (GS) in P+I12 group also showed positive results. Conclusion: Considering all the results together, P+I2 group and P+I12 group showed the best results, with intact retinal tissue structure and the better IF staining results of Pax6 and Rhodopsin. Therefore, when IF staining experiment is planned to be performed on retinae of mice, perfusion combined with immersion for 2 to 12 hours can be used as the optimal tissue fixation method. |
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