In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea.
In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea.
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DOI：
10.1186/1471-213x-13-8
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发表时间：
2013-03-12
影响因子：
--
通讯作者：
Newmark PA
中科院分区：
文献类型：
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作者：
King RS;Newmark PA
淡水真涡虫Schmidtea meditaliphea已成为再生，干细胞和生殖细胞生物学研究的一个强大的模型。整体原位杂交（WISH）和整体荧光原位杂交（FISH）是确定真涡虫基因表达模式的重要方法。虽然许多基因的表达模式已经阐明了使用既定的协议，确定表达模式，特别是低丰度的转录本仍然是一个挑战。我们在这里表明，在甲酰胺中的短漂白步骤显着增强WISH和FISH的信号强度。为了进一步提高信号灵敏度，我们优化了多种抗半抗原抗体的阻断条件，开发了一种硫酸铜淬灭步骤，几乎消除了自发荧光，并通过酪胺信号放大的迭代轮次增强了信号强度。对于再生的真涡虫的FISH，我们采用了热诱导的抗原修复步骤，该步骤提供了成熟组织的透化和再生组织的保存之间的更好的平衡。我们还表明，叠氮化物最有效地淬灭过氧化物酶活性之间的发展循环的FISH实验。最后，我们应用这些修改来阐明一些低丰度转录本的表达模式。我们在这里提出的修改提供了显着的改善，在信号强度和信号灵敏度的WISH和FISH的涡虫。此外，这些修改可能是广泛的实用程序，在其他模式生物的整体安装FISH。
The freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology. Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians. While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge. We show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH. To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification. For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues. We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments. Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts. The modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians. Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms.