Combined method of whole mount and block‐face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization
Combined method of whole mount and block‐face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization
复制标题
整体安装和块面成像的组合方法：从传统原位杂交中获取基因表达模式的 3D 数据
DOI：
--
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Kazuko Koshiba
中科院分区：
文献类型：
--
作者：
Aldy Anindyawan Sutrisno;Wataru Katano;Hayata Kawamura;Yuki Tajika;Kazuko Koshiba
感兴趣基因的时空表达的可视化是分析基因在器官发育中的参与的基本技术。原位杂交 (ISH) 是可视化基因表达的最流行的方法之一。当对切片或整体标本进行传统 ISH 时，基因表达模式以二维 (2D) 显微图像或标本的表面视图表示。为了从传统 ISH 中获取基因表达的 3 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劳动力，因为它需要研究人员收集大量切片，通过 ISH 标记所有切片，并在 3D 重建之前对它们进行成像。在这里，我们提出了一种快速且低成本的 3D 成像方法，可以从传统 ISH 标记的标本创建 3D 基因表达模式。我们的方法结合了整体原位杂交 (ISH) 和相关显微镜和块面成像 (CoMBI)。使用切片机或低温恒温器对整体 ISH 标记的标本进行切片，并对所有块面进行成像并通过 CoMBI 重建 3D 图像。使用我们的方法获取的 3D 数据显示出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，在需要时可以获得切片的二维显微图像。将 2D 显微图像和 3D 数据关联起来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可用于发育生物学领域。
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.
在小鼠胚胎中，心室形态发生过程中心房标志物的下调是不可逆转的。
DOI：
10.1242/dev.125.22.4427
发表时间：
1998
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Gruber,PJ;Kubalak,SW;Chien,KR
通讯作者：
Chien,KR