Identifying Donor Immune Cells in the Intestinal Mucosa of Chimeric Recipient Mice by Immunofluorescence

Murine competitive congenic bone marrow transplantation is an important method used to identify genetic contributions to immune cell abundance, activation, differentiation, and mobilization, either in the bone marrow or in the peripheral organs of the host. This enables analysis of interactions between cells from both donors in relation to each other and the host itself. Flow cytometry is the predominant approach for experimental designs such as these for its powerful quantitative capacity and ability to identify rare and complex immune cells using cell surface markers. However, information regarding immune cell localization and interaction in peripheral organs is usually unexplored due to technical difficulties in identifying immune cells through immunofluorescence, as tissue fixation results in protein cross-linking and inadvertent masking of protein epitopes. In addition, commercially available antibodies validated for flow cytometry are generally designed to interact with integral epitopes expressed on the outer cell membrane. This feature renders conventional immunofluorescence using formalin-fixed tissues impractical because antigen retrieval techniques result in breakage of cross-linked proteins by the denaturation process. Here, we have adapted and improved routinely used immunofluorescence protocols to enable identification of CD45⁺ immune cells and their specific isoforms in the intestinal tissue of recipient mice, permitting tracking of the immune cell origins and localization within peripheral organs. This is achieved by immediately freezing intestinal specimens in OCT and performing methanol fixation only after tissue sectioning, as opposed to formalin-based fixation immediately post-tissue harvest as in conventional immunofluorescence protocols, allowing us to identify immune cell markers using antibodies validated for flow cytometry. This technique adds a new layer of information that allows characterization of how immune cells interact with one another in situ in a mouse model of competitive congenic bone marrow transplant. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence of CD45 isoforms in fresh-frozen colon tissue using flow cytometry-validated antibodies Basic Protocol 2: Processing RGB images to specifically detect each cell type for downstream analysis using Fiji software.