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

Kim C M Jeucken,Jasper J Koning,Jan Piet Van Hamburg,Reina E Mebius,Sander W Tas

doi:10.3389/fimmu.2021.699336

Abstract

Whole mount tissue immunolabeling and imaging of complete organs has tremendous benefits in characterizing organ morphology. Here, we present 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 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 that TNFRII signaling is also to some extent involved in this process as TNFRII-/- PLNs had a 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. Together, these results underline the potential of whole mount tissue staining and advanced imaging techniques to unravel even subtle changes in lymphoid tissue architecture.

Highlights

Microscopic visualization of molecular markers and cells with immunolabeling has been among the most important breakthroughs in biological research
Total peripheral lymph nodes (PLNs) volume calculated based on 3 dimensional (3D) volumetric surface was similar for all genotypes (TNFRI-/-; 2.23 ± 0.311 mm3, or TNFRII-/-; 2.17 ± 0.401, WT; 2.18 ± 0.422)
We visualized and quantified potential changes in B cell clusters of TNFRI-/- and TNFRII-/- PLNs compared to WT mice

Summary

Introduction

Microscopic visualization of molecular markers and cells with immunolabeling has been among the most important breakthroughs in biological research. For a long time microscopic imaging was constrained to tissue sections with a thickness of mere micrometers, but over the years tremendous advantages have been made accumulating in a plethora of techniques for immunolabeling, optical clearing and imaging of complete organs, animals or even human embryos with minimal disassembly of the tissue [1,2,3,4,5]. These methods apply a combination of whole mount immunofluorescence (IF) staining, optical tissue clearing and ultramicroscopy (UM) imaging [1]. These organs include lymph nodes (LNs), which are relatively easy to subject to techniques for 3D visualization and analysis

Methods

Results

Conclusion