Histological study and LYVE-1 immunolocalization of mouse mesenteric lymph nodes with "in vivo Cryotechnique"

The "in vivo cryotechnique" (IVCT) is a powerful tool to directly freeze living animal organs in order to maintain biological components in frozen tissues, reflecting their native states. In this study, mesenteric lymph nodes of living mice were directly frozen with IVCT, and we did morphological studies and immunohistochemical analyses on a hyaluronic acid receptor, LYVE-1. In lymph nodes, widely open lymphatic sinuses were observed, and many lymphocytes adhered to inner endothelial cells along subcapsular sinuses. The LYVE-1 was clearly immunolocalized at inner endothelial cells of subcapsular sinuses, as well as those of medullary sinuses. Conventional pre-embedding electron microscopy also showed LYVE-1 immunolocalization along both the apical and basal sides of cell membranes of inner endothelial cells. By triple-immunostaining for LYVE-1, smooth muscle actin, and type IV collagen, the LYVE-1 was immunolocalized only in the inner endothelial cells, but not in outer ones which were surrounded by collagen matrix and smooth muscle cells. Thus, the functional morphology of lymph nodes in vivo was demonstrated and LYVE-1 immunolocalization in inner endothelial cells of subcapsular sinuses suggests hyaluronic acid incorporation into lymph node parenchyma.