Playback is 10 structures/s. FDCs (19). A stromal cell subset, CXCL12-expressing reticular cells (CRCs), is usually localized to the paracortical side of the follicles and upon GC formation, provides functional support for the dark zone (20, 21). Most recently, Cyster and colleagues showed further heterogeneity in FSCs through single-cell RNA sequencing analysis (22), even though functional significance of such highly diversified FSCs remains obscure. The anatomical region ranging from the deep cortex to the medulla of the LN is usually presumably important for innate and adaptive responses given the localization of a variety of immune cells including macrophages, NK cells, and plasma cells (23C27). However, knowledge of this area is limited; the indistinct distribution of immune cells, as compared to the cortex, and the intricate structure of intertwined blood vessels and lymphatic Maxacalcitol sinuses could have hampered in-depth studies. The characteristic anatomies in this area suggest the presence of functionally unique stromal cells. In this study, we sought to clarify the relevance of FSCs for the arrangement of LN subcompartments by utilizing several gene reporters expressed in stromal compartments. This led to the discovery of a novel FSC type that supports an area in the deep cortex, which was unique from FSCs in the T cell area as well as the medulla. These observations bring about a comprehensive view of multi-layered subcompartments and associated FSC subsets in the LN. Materials and methods Mice C57BL/6JJcl and BALB/cAJcl-mice were purchased from CLEA, Japan. B6.129P2-(mouse strain (RBRC04200) was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. Mice were managed and crossed under specific pathogen-free Maxacalcitol conditions in the animal facility of Niigata University or college. All animal procedures were approved by the Maxacalcitol Committee on Animal Research at Niigata University or college. Generation of reporter mice Genomic fragments of the gene locus were amplified from RENKA ES cell genomic DNA by PCR. The targeting vector was constructed as follows: the second exon of was inserted with an in-frame start codon followed by the gene encoding EYFP (venus), an internal ribosomal access site (IRES), the gene encoding CreERT2, and in reverse orientation, a FRT-flanked neomycin resistance gene (neor) cassette. The linearized targeting construct was electroporated into RENKA B6 mouse ES cells and G418 resistant colonies were screened by Southern blotting using AflII- or HindIII-digested genomic DNA using a neor-flanking probe. Targeted ES clones were injected into B6 blastocysts and chimeras were mated to B6 mice. Targeted alleles were screened by PCR using the primers: 5-CTTGTCTGGTCTGCATTTCTTGGC-3 (sense; PDGFR-gF); 5-TGAACTTGTGGCCGTTTACGTCG-3 (antisense; EGFP-R10). Antibodies The following fluorochrome-conjugated, biotin-conjugated, or unconjugated main antibodies were purchased: anti-CD3e (145-2C11), anti-B220 (RA3-6B2), anti-CD11c (N418), anti-F4/80 (BM8), anti-CD45 (30-F11), anti-CD31 (390), and anti-podoplanin (8.1.1) (eBioscience); anti-desmin (Abcam); ER-TR7 (BMA); anti-CD35 (8C12), anti-IgDb (217-170), and anti-CD138 (281-2) (BD Biosciences); anti-VCAM-1 (BAF643), anti-RANKL (BAF462), anti-CXCL13 (BAF470), anti-LYVE-1 (BAF2125), anti-LepR (BAF497) (R&D Systems); anti-laminin (LSL); anti-GFP and anti-RFP (MBL). For secondary reagents, PE-, APC-, AlexaFluor488-, 546-, 555-, 594-, or 633-conjugated streptavidin, anti-rabbit IgG, and anti-rat IgG were purchased from Molecular Probes. Circulation cytometry Single-cell suspensions were prepared from superficial LNs (cervical, axillary, brachial, inguinal, and popliteal) through digestion with 1 mg/mL collagenase D and 0.1 mg/mL DNase I (Roche Diagnostics) as explained (32), and stained with anti-CD45, anti-CD31, and anti-gp38/podoplanin antibodies and propidium iodide. Data were acquired using Maxacalcitol a FACSCalibur (BD Biosciences) circulation cytometer and analyzed with CellQuest (BD Biosciences) or FlowJo. Immunohistochemistry Isolated LNs (inguinal, brachial, cervical, and popliteal) were fixed with 0.05% phosphate C13orf18 buffer containing 0.075 M L-lysine (pH 7.4), 0.01 M NaIO4, and 1% paraformaldehyde (PLP fixative) at 4C for 16C24 h. After fixation, LNs were equilibrated gradually with 10, 20, and 30% sucrose in PBS at 4C, embedded in OTC compound (Sakura Finetechnical), and frozen at ?80C. Frozen sections (10 m) were made using a cryostat (Leica Biosystems) and post-fixed with chilly acetone for 3 min. To properly evaluate the pattern of subcompartments and the location of FSC subsets, we made LN sections that incorporated the cortexCmedulla axis. Sections.