AUTHOR=Jeucken Kim C. M. , Koning Jasper J. , van Hamburg Jan Piet , Mebius Reina E. , Tas Sander W. 

TITLE=A Straightforward Method for 3D Visualization of B Cell Clusters and High Endothelial Venules in Lymph Nodes Highlights Differential Roles of TNFRI and -II

JOURNAL=Frontiers in Immunology

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.699336

DOI=10.3389/fimmu.2021.699336

ISSN=1664-3224

ABSTRACT=Whole mount tissue immunolabeling and imaging of complete organs has tremendous benefits in characterizing organ morphology. Here, we set up a straightforward method for immunostaining, clearing and imaging of whole murine peripheral lymph nodes (PLNs) for detailed analysis of their architecture and discuss all procedures in great detail in a step-by-step approach. Given the importance of tumor necrosis factor receptor (TNFR) signaling in development of PLNs we used TNFRI-/- and TNFRII-/- mice models as proof-of-concept for this technique by visualizing and analyzing structural changes in PLN B cell clusters and high endothelial venules (HEVs). Samples were subjected to de- and rehydration with methanol, labeled with antibodies for B cells, T cells and high endothelial venules (HEVs) and optically cleared using benzyl alcohol-benzyl benzoate. Imaging was done using LaVision light sheet microscope and analysis with Imaris software. Using these techniques, we confirmed previous findings that TNFRI signaling is essential for formation of individual B cell clusters. In addition, our data suggest a formerly unknown role for TNFRII signaling in this process since TNFRII-/- PLNs had B cell cluster morphology reminiscent of TNFRI-/- PLNs. Moreover, visualization and objective quantification of the complete PLN high endothelial vasculature unveiled reduced volume, length and branching points of HEVs in TNFRI-/- PLNs, revealing an earlier unrecognized contribution of TNFRI signaling in HEV morphology. These results would be missed using conventional histology methods thereby underlining the potential of the described whole mount tissue staining and imaging techniques to unravel (subtle) changes in architecture.