Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.

微生物对宿主细胞的影响取决于微生物的特性、它们的空间定位以及它们对特定宿主细胞群的反应。以空间解析方式对微生物和宿主细胞进行多模态分析将有助于研究天然组织环境中这些复杂的相互作用，可能是在临床标本中。虽然保存每个微生物和宿主细胞隔室的技术已被用于分别检查组织和微生物，但我们努力开发同时分析这两个隔室的方法。在此，我们建立了使用泊洛沙姆 407（也称为 Pluronic F-127）保存粘液的原始方法，泊洛沙姆 407 是一种具有粘液粘附特性的热可逆聚合物。我们证明，与使用传统福尔马林固定石蜡包埋 (FFPE) 管道处理组织时明显不存在的情况相比，这种方法可以保留结肠中粘液双层的空间定义隔室和其中的细菌群落。此外，宿主细胞抗体染色的抗原被保留，16S rRNA 荧光原位杂交 (FISH) 的信号强度在泊洛沙姆固定的样品中得到增强。这反过来使我们能够使用修改后的多重免疫荧光 (MxIF) 协议集成多模式分析。重要的是，我们配制了泊洛沙姆 407，可在室温下聚合和交联，以用于临床工作流程。