From image to insight: optimized multiplex staining panel for formalin-fixed, paraffin-embedded mouse bone marrow enables spatial profiling of the hematopoietic niche.

Cellular composition and spatial architecture of the bone marrow niche play a critical role in regulating both normal and malignant hematopoiesis. Although flow cytometry enables rapid analysis of hematopoietic and niche cell composition, it requires single-cell dissociation, thereby disrupting the spatial context. In contrast, immunofluorescence imaging preserves tissue architecture but is limited in the number of markers that can be simultaneously detected. Recent advances in multiplex staining technologies now permit visualization of numerous antigens within a single tissue section, providing a more comprehensive view of the bone marrow microenvironment. Despite the central role of murine models in studying hematopoiesis, validated multiplex panels for analyzing murine bone marrow remain limited. This is particularly true for formalin-fixed, paraffin-embedded (FFPE) sections, where challenges such as high autofluorescence and tissue damage during antigen retrieval place substantial demands on tissue preparation. In this study, we optimized tissue preparation for staining murine FFPE bone marrow and developed an efficient multiplex immunohistochemistry panel for detecting hematopoietic stem cell-containing populations within their native niche architecture. Applying our panel to FFPE bone marrow sections from Jak2V617F mice revealed alterations in the megakaryocytic niche, demonstrating the utility of this approach for characterizing disease-associated spatial changes within the hematopoietic microenvironment.