Abstract：
Staining frozen sections is often required to distinguish cell types for spatial transcriptomic studies of the brain. The impact of the staining methods on the RNA integrity of the cells becomes one of the limitations of spatial transcriptome technology with microdissection. However, there is a lack of systematic comparisons of different staining modalities for the pretreatment of frozen sections of brain tissue as well as their effects on transcriptome sequencing results. In this study, four different staining methods were analyzed for their effect on RNA integrity in frozen sections of brain tissue. Subsequently, differences in RNA quality in frozen sections under different staining conditions and their impact on transcriptome sequencing results were assessed by RNA-seq. As one of the most commonly used methods for staining pathological sections, HE staining seriously affects the RNA quality of frozen sections of brain tissue. In contrast, the homemade cresyl violet staining method developed in this study has the advantages of short staining time, low cost, and less RNA degradation. The homemade cresyl violet staining proposed in this study can be applied instead of HE staining as an advance staining step for transcriptome studies in frozen sections of brain tissue. In the future, this staining method may be suitable for wide application in brain-related studies of frozen tissue sections. Moreover, it is expected to become a routine step for staining cells before sampling in brain science.
摘要：
对于大脑的空间转录组学研究，通常需要染色冷冻切片来区分细胞类型。染色方法对细胞RNA完整性的影响成为具有显微解剖的空间转录组技术的限制之一。然而,对于脑组织冰冻切片预处理的不同染色方式及其对转录组测序结果的影响，缺乏系统的比较。在这项研究中,分析了四种不同的染色方法对脑组织冷冻切片中RNA完整性的影响。随后，通过RNA-seq评估不同染色条件下冷冻切片中RNA质量的差异及其对转录组测序结果的影响。作为病理切片最常用的染色方法之一,HE染色严重影响脑组织冰冻切片的RNA质量。相比之下,本研究开发的自制甲酚紫染色方法具有染色时间短的优点,低成本,和较少的RNA降解。本研究中提出的自制甲酚紫染色可以代替HE染色作为脑组织冷冻切片转录组研究的高级染色步骤。在未来,这种染色方法可能适用于与大脑相关的冷冻组织切片研究。此外，它有望成为脑科学中采样前染色细胞的常规步骤。
