The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue, as local interactions precipitate intra-cellular events that often lead to changes in expression. However, our understanding of these processes in tissues, whether healthy or diseased, is limited at present owing to the difficulty in acquiring comprehensive transcriptional programs of spatially- and phenotypically-defined cells in situ. Here we present a robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved transcriptional programs with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues sufficient to resolve even isoforms. The protocol is optimized to protect the RNA with a small molecule inhibitor, the ribonucleoside vanadyl complex (RVC), which thereby enables the typical time-consuming immunostaining and laser capture steps of this procedure during which RNA is usually severely degraded in existing approaches. The efficacy of this approach is exemplified by the characterization of differentially expressed genes between the mouse small intestine lacteal cells at the tip versus the main capillary body, including those that function in sensing and responding to local environmental cues to stimulate intra-cellular signalling. With the extensive repertoire of specific antibodies that are presently available, our method provides an unprecedented capability for the analysis of transcriptional networks and signalling pathways during development, pathogenesis, and aging of specific cell types within native tissues.

组织的功能从根本上不仅取决于组成细胞的表型，还取决于它们在组织中的空间组织，因为局部相互作用会促成细胞内事件，这些事件通常会导致表达变化。然而，由于难以获得空间和表型定义细胞的综合转录程序，我们对组织中这些过程的理解，无论是健康的还是患病的，目前都是有限的。原位. 在这里，我们提出了一种基于免疫荧光引导的激光捕获显微切割 (immuno-LCM-RNAseq) 的稳健方法，该方法可以从速冻或 RNAlater 处理的临床组织中获取精确解析的转录程序，其中的细胞数量少至足以解析亚型。该协议经过优化，以使用小分子抑制剂核糖核苷钒复合物 (RVC) 保护 RNA，从而实现该过程中典型的耗时免疫染色和激光捕获步骤，在此过程中，RNA 通常在现有方法中被严重降解。这种方法的功效体现在小鼠小肠乳突细胞尖端与毛细血管主体之间差异表达基因的表征，包括那些能够感知和响应局部环境线索以刺激细胞内信号传导的功能。凭借目前可用的大量特定抗体，我们的方法为分析天然组织内特定细胞类型的发育、发病机制和衰老过程中的转录网络和信号通路提供了前所未有的能力。