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

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.

Nucleolar Architecture Is Modulated by a Small Molecule, the Inositol Pyrophosphate 5-InsP7

“…The nucleolar signals were captured using the Z-stack function (5 × 1 µm Z-slices). The 3D rendering and volume measurements were performed using the SURFACE function in Imaris software [34,35].…”

3D imaging and morphometric descriptors of vascular networks on optically cleared organs

“…Scientific literature survey shows that the majority of the articles on 3D analysis of vascular networks in optically cleared organs use commercial software, allowing the automatic segmentation of the vascular network, regardless of the thresholding technique associated. 4 , 5 , 13 , 14 In addition with commercial software, some laboratories have developed their own segmentation algorithms or computational tools to extract and quantify vasculatures. 6 , 15 , 16 , 17 Finally, around fifteen thresholding techniques are available with the open-source freeware ImageJ, belonging to 3 categories of the Sezgin and Sankur’s classification, based on 1 the shape of the pixel gray-level histogram (the properties of the gray-level histogram such as its peaks and valleys), (2) the clustering analysis of the pixel gray-level histogram (the gray-level histogram is considered as corresponding to two clusters of pixels representing the object and the background), and (3) the entropy of the pixel gray-levels of the image (the image is considered as composed of “information” associated to the signal but mainly of noise, with a certain degree of uncertainty on which gray-level correspond to what).…”

“…A survey of the literature on optical clearing and 3D imaging of vascular networks indicates that the most of the articles use, for the segmentation step, Imaris software (Bitplane, Switzerland) FilamentTracer function. It has have been used to isolate the vascular network in several organs such as the heart, 4 the liver and the lung, 5 the pancreas, 13 the brachial and inguinal skeletal muscles, 14 and the kidney. 9 Imaris is a commercial software allowing 3D and 4D image analysis, and FilamentTracer is an extension initially allowing the automatic tracing of neurons and the detection of their spines, but extended for the tracing of any filamentous structures as blood vessels.…”

“… 3 Total length (μm) 13,000,000 Cap LV B6J Lectin IV – iDisco+ LSM ImageJ Nicolas and Roux 19 11,500,000 Cap S B6J Lectin IV – iDisco+ LSM ImageJ Nicolas and Roux 19 7,110,000 Cap RV B6J Lectin IV – iDisco+ LSM ImageJ Nicolas and Roux 19 27,000,000 Cap LV B6J Lectin IV – iDisco+ CM ImageJ Nicolas and Roux 19 1,000,000 HEVs PLNs B6J MECA-79 AB - BABB LSM Imaris Jeucken et al. 14 3,000 Cap IPIs NOD Lectin IV – No OC CM Imaris El-Gohary et al. 13 23,000 BVs Retina B6J CD31 AB - EyeCi LSM Imaris Henning et al.…”

Splenic Architecture and Function Requires Tight Control of Transmembrane TNF Expression

“…The observed defects seem mainly dependent on TNFRI signaling, as mice lacking TNFRI have an abnormal SLO morphology, which is comparable to TNF −/− mice. This morphology includes aberrant architecture of lymphoid follicles, as mice lacking TNFRI have a peripheral B cell sheet rather than individual lymphoid follicles [7,8]. We recently demonstrated that PLNs (brachial and inguinal) of TNFRII −/− mice have a morphology reminiscent of TNFRI −/− mice, since PLNs of these mice have decreased individual lymphoid follicles, suggesting a previously unknown role for TNFRII signaling in SLO architecture [8].…”