File:Topological-Small-World-Organization-of-the-Fibroblastic-Reticular-Cell-Network-Determines-Lymph-pbio.1002515.s007.ogv

English: FRC network 3-D reconstruction and analysis pipeline. Confocal microscopy analysis was performed on whole LN histological sections of naive adult Ccl19eyfp mice stained for EYFP, PDPN, and DAPI. One to two T cell zones (approximately 300 x 300 x 30 μm) per LN were acquired in high resolution in order to generate the representative T cell zone FRC network. A small zoom-in area with several single FRCs was selected for visualization purposes. The cell body was stained by EYFP, the cell protrusions were accurately visualized by PDPN, and DAPI staining was used to identify cell nuclei. In order to identify single FRCs, the 3-D reconstructions of EYFP+ FRCs (white) were masked to the DAPI channel. The whole EYFP+ network was then 3-D reconstructed using an automatic threshold, and the surface area and volume of the whole FRC network was calculated. FRCs suitable for single-cell analysis (yellow) were selected and their morphological parameters were determined (e.g., single cell surface area, volume, and sphericity). Centers of homogeneous mass of FRCs were determined based on the 3-D reconstructions and selected as nodes for topological analysis. The FRC network edges (connections) were traced based on the physical connections between neighboring FRCs, and an undirected, unweighted network graph was generated. The adjacency matrix of the FRC network containing connectivity information was created from the network graph and imported into RStudio for subsequent topological network analysis.