Abstract：
The red flour beetle, Tribolium castaneum, is an emerging model system well suited to the study of embryonic brain development and evolution (see Chapters 11 and 13 ). Brain genesis is driven by specific gene products whose expression underlies a tight spatiotemporal control. Therefore, the analysis of gene expression in time and space provides valuable insights into the molecular mechanisms that govern brain development. Since Tribolium-specific antibodies are scarce, fluorescent RNA in situ hybridization is the method of choice to determine the dynamics of individual gene expression. We have modified common RNA in situ protocols to facilitate the concomitant detection of two gene-specific expression patterns (double fluorescent RNA in situ). In addition, we describe a procedure which combines fluorescent single RNA in situ and immunostaining with gene-specific antibodies. Conventional in situ using RNA probes that are complementary to mature mRNAs often produce diffuse signals. We demonstrate that RNA in situ probes complementary to intronic gene sequences facilitate single cell resolution because the fluorescent signal is restricted to the nucleus. We believe our protocols can be adapted easily to suit the analysis of brain development in other insect species.
摘要：
红面粉甲虫,蓖麻,是一个新兴的模型系统，非常适合研究胚胎大脑发育和进化（参见第11章和第13章）。脑发生是由特定基因产物驱动的，其表达是严格的时空控制的基础。因此,对基因表达在时间和空间上的分析为控制大脑发育的分子机制提供了有价值的见解。由于Tribolium特异性抗体很少，荧光RNA原位杂交是确定单个基因表达动力学的首选方法。我们已经修改了常见的RNA原位方案，以促进同时检测两种基因特异性表达模式（原位双荧光RNA）。此外,我们描述了一种将荧光单RNA原位和免疫染色与基因特异性抗体相结合的方法。常规原位使用与成熟mRNA互补的RNA探针通常产生扩散信号。我们证明，与内含子基因序列互补的RNA原位探针有助于单细胞分辨率，因为荧光信号仅限于细胞核。我们相信我们的协议可以很容易地适应其他昆虫物种的大脑发育分析。
