Mesoscopic examination of naive PLN volume, B-cell follicle structure, and HEV network. (A) Workflow of OPT analysis of PLN architecture. PLN labeled with B220-Alexa488 and MECA-79-Alexa594 mAbs were dehydrated, cleared in BABB, and imaged in an OPT scanner as described in “Whole-mount staining for secondary lymphoid organs and preparation for OPT” (top). In some experiments, MECA-79-Alexa568 was used with similar results. After OPT image acquisition, samples can be further analyzed by high-resolution laser scanning microscopy (LSM) or rehydrated for flow cytometry analysis (bottom). As seen on the left (OPT), the current resolution clearly identifies HEV but not individual endothelial cells as seen when high-resolution LSM is used. Scale bar in LSM = 40 μm. (B) Schematic representation of primary OPT images (left; shown is 1 of 400 raw images before 3-dimensional [3D] reconstruction), 3D reconstruction (middle), and quantification of fluorescently labeled structures (right). Before OPT imaging, PLNs were whole-mount labeled with B220-Alexa488 (for B-cell follicles) and MECA-79-Alexa594 (anti-PNAd for HEV) mAbs and processed as described in “OPT scanning and software-based 3D reconstruction and quantification.” The length of the long axis of the inguinal PLN shown is 2.4 mm.