共聚焦和荧光显微镜用于定量 RNAscope 和免疫荧光图像的比较。
A comparison of confocal and epifluorescence microscopy for quantification of RNAScope and immunohistochemistry fluorescent images.
机构信息
The Center for Voice and Swallowing, Department of Otolaryngology-Head & Neck Surgery, Columbia University Irving Medical Center, New York, NY, United States.
出版信息
J Neurosci Methods. 2024 Mar;403:110050. doi: 10.1016/j.jneumeth.2023.110050. Epub 2023 Dec 23.
BACKGROUND
Quantification of RNA expression and protein production in fluorescent stainings provides critical information concerning neurodevelopment. A trustable independent quantification technique requires acquisition of reliable images prior to image processing. There is uncertainty in existing literature regarding the use of confocal microscopy compared to standard epifluorescence microscopy, especially in the context of RNA in situ hybridization protocols.
NEW METHOD
The hindbrains of developing rat embryos from embryologic day 14 (E14) to E20 were sectioned and stained for expression of Hoxb1, Hoxb2, and Phox2b using both RNAScope and immunohistochemistry. Islet1 was used for identification of hindbrain motoneuron cell bodies. Slides were imaged using both confocal and epifluorescence microscopy.
RESULTS
Expression patterns of both mRNA and protein were similar in both imaging modalities. Analyses of Hoxb1 and Hoxb2 mRNA expression were particularly concordant between-scopes, with similar p-values and posthoc differences between timepoints. Confocal imaging of Hoxb2 protein yielded a significant peak at E18, but this level of significance was not reached using epifluorescence microscopy. Although similar trends were observed, only Phox2b RNAScope results were statistically significant when analyzed with confocal microscopy. In contrast, Phox2b immunostaining analyses showed significant differences using both microscopes.
COMPARISON WITH EXISTING METHODS
Researchers may save time and financial resources if epifluorescence microscopy provides comparable or equal results as confocal.
CONCLUSIONS
Epifluorescence microscopy appears sufficient for quantification of RNAScope experiments with relatively low puncta per cell, while confocal microscopy gives clearer definition to immunohistochemical protein relationships and may be preferable especially in targets with low protein production.
背景
荧光染色中 RNA 表达和蛋白质产生的定量提供了与神经发育相关的关键信息。在进行图像处理之前，获取可靠的图像是一种可靠的独立定量技术所必需的。在 RNA 原位杂交方案方面，与标准荧光显微镜相比，使用共聚焦显微镜存在不确定性，尤其是在现有文献中。
新方法
使用 RNAScope 和免疫组织化学对来自胚胎发育第 14 天（E14）到 E20 的发育中大鼠胚胎的后脑进行染色，以表达 Hoxb1、Hoxb2 和 Phox2b。Islet1 用于鉴定后脑运动神经元细胞体。使用共聚焦和荧光显微镜对载玻片进行成像。
结果
两种成像方式的 mRNA 和蛋白质表达模式相似。两种方法的 Hoxb1 和 Hoxb2 mRNA 表达分析特别一致，具有相似的 p 值和时间点之间的事后差异。Hoxb2 蛋白的共聚焦成像在 E18 时产生了一个显著的峰值，但使用荧光显微镜未达到该水平的显著性。尽管观察到相似的趋势，但只有 Phox2b RNAScope 结果在使用共聚焦显微镜进行分析时具有统计学意义。相比之下，Phox2b 免疫染色分析在两种显微镜下均显示出显著差异。
与现有方法的比较
如果荧光显微镜提供与共聚焦相似或相等的结果，研究人员可能会节省时间和财务资源。
结论
对于相对每细胞 puncta 较少的 RNAScope 实验，荧光显微镜似乎足以进行定量，而共聚焦显微镜可以更清晰地定义免疫组织化学蛋白关系，尤其是在蛋白质产量较低的目标中，可能更可取。
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