Semi-Automated Analysis of Lymph Node Immune and Fibroblast Architecture From Immunofluorescent Images

Tissues are highly ordered structures, with cellular localization essential to the optimal function of the tissue. In lymph nodes the spatial arrangement of immune cells and their associated with stromal cell architecture is essential for the generation of optimal immune responses. Circumstances such as aging and chronic disease can alter lymph node architecture in a manner that impacts immune function. Immunologists have developed many tools for analyzing the cellular content of lymphoid tissue, with flow cytometry a central workhorse in the field. While flow cytometry is a powerful high-throughput tool for analyzing multiple cell types in a tissue, it cannot assess where those cells are located. Imaging, on the other hand, is relatively low throughput, with objective image analysis often presenting a significant hurdle for many immunologists. Here we describe two analysis tools built in Fiji (ImageJ) that semi-automate the analysis of lymph node spatial architecture, can be adapted to different antibody staining panels, and are straightforward to implement with little computational experience. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Quantifying changes in lymph node regional architecture Alternate Protocol: Adapting KVA1 for use on spleen tissue sections Basic Protocol 2: Measuring follicular dendritic cell network area within individual B cell follicles.