Combined method of whole mount and block‐face imaging: Acquisition of <scp>3D</scp> data of gene expression pattern from conventional in situ hybridization
Combined method of whole mount and block‐face imaging: Acquisition of <scp>3D</scp> data of gene expression pattern from conventional in situ hybridization
复制标题
整体贴片和块面成像相结合的方法：从常规原位杂交中获取基因表达模式的<scp>3D</scp>数据
DOI：
10.1111/dgd.12827
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Koshiba‐Takeuchi Kazuko
中科院分区：
文献类型：
--
作者：
Sutrisno Aldy Anindyawan;Katano Wataru;Kawamura Hayata;Tajika Yuki;Koshiba‐Takeuchi Kazuko
感兴趣的基因的时空表达可视化是分析基因参与器官发育的基本技术。原位杂交（ISH）是目前最流行的基因表达可视化方法之一。当传统的ISH在切片或整片标本上进行时，基因表达模式在2维（2D）显微镜图像或标本表面视图中表示。为了从传统的ISH中获得基因表达的三维（3D）数据，传统上采用“连续切片法”。然而，这种方法需要大量的时间和人力，因为它需要研究人员收集大量的切片，用ISH标记所有切片，并在3D重建之前对其进行成像。在这里，我们提出了一种快速、低成本的3D成像方法，可以从传统的ISH标记的标本中创建3D基因表达模式。我们的方法包括全贴装ISH和相关显微镜和块面成像（CoMBI）的组合。用切片机或低温恒温器切片整片ISH标记的标本，所有块面成像，并使用CoMBI重建3D图像。用我们的方法获得的三维数据显示了足够的质量来分析发育中的小鼠心脏的形态和基因表达模式。此外，在需要时，还可以获得切片的二维显微图像。将二维显微图像和三维数据相关联可以帮助注释基因表达模式并了解发育器官的解剖结构。这些结果表明我们的方法在发育生物学领域有一定的应用价值。
Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole‐mount specimens, the gene expression pattern is represented in 2‐dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3‐dimensional (3D) data of gene expression from conventional ISH, the “serial section method” has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low‐cost 3D imaging method that can create 3D gene expression patterns from conventional ISH‐labeled specimens. Our method consists of a combination of whole‐mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole‐mount ISH‐labeled specimens were sliced using a microtome or cryostat, and all block‐faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.