Spatial Distribution and Characterization of Mouse Lymphoid Tissue Using Fully Automated Multiplex Immunofluorescence on COMETTM 3909

Description Spatial biology is rapidly emerging as a powerful tool for deepening our knowledge of the complex, dynamic nature of tissue-specific immune responses. It involves investigating the interactions between lymphoid cells, antigen-presenting cells, and non-lymphoid cells within tissues. Multiplex immunohistochemistry (IHC) and immunofluorescence (IF) are ideal techniques for characterizing the spatial aspects of immune responses. To streamline accurate marker detection, emerging automated staining and imaging platforms require qualified antibodies with platform-specific and proven specificity and sensitivity. Using IHC antibodies from R&D SystemsTM, we used a systematic process to qualify antibodies for use on the Lunaphore COMET™ platform in murine lymphoid tissue. COMETTM offers a novel sequential immunofluorescence (seqIF™) approach that utilizes cycles of staining, imaging, and elution with unconjugated primary antibodies and fluorescently labeled secondary antibodies. In this study, we distinguish important cell types, including pan-cytokeratin+ epithelial cells, CD45+ hematopoietic cells, and CD31+ endothelial cells. Simultaneously, we can further characterize hematopoietic cells using phenotypic markers, including Ki67, the regulatory transcription factor FoxP3, and the activation marker CD86. This approach provides a powerful platform for characterizing tissue-specific immune responses, differentiating disease states, and identifying potential therapeutic targets.