Confocal-Compatible Workflow for Sectioning, Staining, and Imaging Serial Vibratome Sections for 3D Anatomical Reconstruction of the Lymph Node.

3D anatomical reconstruction provides quantitative insight into tissue architecture, variability, and function. However, existing whole-organ imaging methods, such as light-sheet microscopy and micro-CT, are often costly, slow, and inaccessible for many laboratories. We present an accessible, confocal-compatible workflow for harvesting, sectioning, and staining murine lymph nodes for high-fidelity 3D reconstruction. The protocol preserves Z-plane resolution through vibratome sectioning and immunostaining, enabling detailed visualization of lymph node lobules and vasculature using standard confocal microscopes. The resulting image stacks can be used for quantitative morphometry and spatiotemporal pharmacokinetic modeling. This reproducible workflow expands access to spatial and structural studies of the lymph node and can be adapted to other small, heterogeneous organs.