Journal: Respiratory Research

Article Title: Increased number and altered phenotype of lymphatic vessels in peripheral lung compartments of patients with COPD

doi: 10.1186/1465-9921-14-65

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry

Article Snippet: After incubation with antibodies against goat anti-human CCL21 or mouse anti-human D6, sections were incubated with biotinylated rabbit anti-goat (1:200, BA-1000, Vector Laboratories, Inc., Burlingame, CA, USA) or biotinylated horse anti-mouse IgG secondary antibodies (1:200, BA-2000, Vector Laboratories).

Techniques:

Journal: Respiratory Research

Article Title: Increased number and altered phenotype of lymphatic vessels in peripheral lung compartments of patients with COPD

doi: 10.1186/1465-9921-14-65

Figure Lengend Snippet: Increased number of alveolar CCL21- and D6-positive lymphatic vessels in patients with advanced COPD. (A) Percentage of CCL21-positive lymphatic vessels among all Prox1-positive lymphatics and (B) number of CCL21-positive lymphatic vessels in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma in never smokers and patients with GOLD stage IV COPD. (C) Percentage of D6-positive lymphatic vessels among all Prox1-positive lymphatics and (D) number of D6-positive lymphatic vessels in similar lung compartments as for (A and B) in never smokers and patients with GOLD stage IV COPD. Mann–Whitney rank sum test was used for comparison between two groups. Horizontal lines indicate median value for each cohort. ** p < 0.01. (E and F) Immunohistochemical staining for CCL21 (red) and Prox1 (brown nuclei) and (G) immunohistochemical staining for D6 (red) and Prox1 (brown nuclei) in sections of patients with GOLD stage IV COPD. LA, lymphoid aggregate. Sections were counterstained with Mayer’s haematoxylin (blue stain). Scale bars: (E , G) 20 μm; (F) 30 μm.

Article Snippet: After incubation with antibodies against goat anti-human CCL21 or mouse anti-human D6, sections were incubated with biotinylated rabbit anti-goat (1:200, BA-1000, Vector Laboratories, Inc., Burlingame, CA, USA) or biotinylated horse anti-mouse IgG secondary antibodies (1:200, BA-2000, Vector Laboratories).

Techniques: MANN-WHITNEY, Immunohistochemical staining, Staining

Journal: Respiratory Research

Article Title: Increased number and altered phenotype of lymphatic vessels in peripheral lung compartments of patients with COPD

doi: 10.1186/1465-9921-14-65

Figure Lengend Snippet: Increased immunoreactivity for CCL21 and D6 in lymphatic endothelium in patients with advanced COPD. (A) Accumulated lymphatic immunoreactivity of CCL21 and (B) D6 in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma in never smokers and patients with GOLD stage IV COPD. Each point indicates the total amount of lymphatic immunoreactivity per unit area. (C) Immunoreactivity of CCL21 and (D) D6 per lymphatic vessel endothelial length in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma. (E and F) Scatter graphs representing the immunoreactivity for (E) CCL21 and (F) D6 in each analysed lymphatic vessel in (C and D) . Mann–Whitney rank sum test was used for comparison between two groups. Horizontal lines indicate median value for each cohort. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After incubation with antibodies against goat anti-human CCL21 or mouse anti-human D6, sections were incubated with biotinylated rabbit anti-goat (1:200, BA-1000, Vector Laboratories, Inc., Burlingame, CA, USA) or biotinylated horse anti-mouse IgG secondary antibodies (1:200, BA-2000, Vector Laboratories).

Techniques: MANN-WHITNEY

Journal:

Article Title: CCL21 Expression Pattern of Human Secondary Lymphoid Organ Stroma Is Conserved in Inflammatory Lesions with Lymphoid Neogenesis

doi: 10.2353/ajpath.2007.061275

Figure Lengend Snippet: CCL21-producing endothelial structures in rheumatoid synovitis display lymphatic features. In situ hybridization (A) and IHC (B) for CCL21 in parallel tissue sections of rheumatoid synovial membrane demonstrate co-localization between CCL21 mRNA (A, in black) and protein (B, in red) in a subset of vascular structures. C–G: A synovial lymphoid aggregate is shown on serial tissue sections immunostained for CCL21 (C, in red), CD31 (D, in red), CD34 (E, in red), PNAd (F, in red), and LYVE-1 (G, in red). Arrows indicate the same vascular structures analyzed in parallel sections. Note that all CCL21-positive vessels (black arrows) are LYVE-1-positive lymphatic vessels. H–K: IHC on sequential sections of a synovial hypertrophic villous showing a subset of vascular structures immunostained for CCL21 (H, in red), LYVE-1 (I, in red), podoplanin (J, in red), and D6 (K, in red). All CCL21-positive vessels (H) are LYVE-1-positive (I) podoplanin-positive (J), and D6-positive (K). The synovial vessel delimited by the black square in H–K is shown at high magnification in L–O, demonstrating the co-localization of CCL21 (L), LYVE-1 (M), podoplanin (N), and D6 (O). P–S: High-magnification views of serial sections of tonsil showing endothelial co-localization of CCL21 (P, in red) with LYVE-1 (Q, in red), podoplanin (R, in red), and D6 (S, in red). T: Single color separations and color merges of double IF staining for CCL21 (in green) and podoplanin (in red) on rheumatoid synovium, showing the co-localization of the two markers. Counterstaining with DAPI in blue. Original magnifications: ×400 (A–G); ×200 (H–K); ×1000 (L–T).

Article Snippet: After TBS washing and an additional blocking in 10% human serum diluted in TBS, goat anti-human CCL21 was applied, followed by incubations with donkey anti-goat IgG biotinylated (Jackson ImmunoResearch Laboratories) and rhodamine Red-X-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: In Situ Hybridization, Staining

Journal:

Article Title: CCL21 Expression Pattern of Human Secondary Lymphoid Organ Stroma Is Conserved in Inflammatory Lesions with Lymphoid Neogenesis

doi: 10.2353/ajpath.2007.061275

Figure Lengend Snippet: The lymphatic nature of CCL21+ endothelial structures is a shared feature of different human chronic inflammatory diseases. Serial immunostaining for CCL21 (A, C, E, and G; in red) and LYVE-1 (B, D, F, and H; in red) is shown on sections of lichen planus (A and B), SS (C and D), UC (E and F), and CD (G and H). All CCL21-positive vessels are LYVE-1-positive. Black arrows indicate the same vascular structures analyzed in parallel sections. Note that CCL21-expressing vessels are LYVE-1-positive both in tissue areas characterized by dense immune-cell infiltration with lymphoid aggregates (E and F) and in noninfiltrated areas (G and H). Original magnifications, ×200.

Article Snippet: After TBS washing and an additional blocking in 10% human serum diluted in TBS, goat anti-human CCL21 was applied, followed by incubations with donkey anti-goat IgG biotinylated (Jackson ImmunoResearch Laboratories) and rhodamine Red-X-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Immunostaining, Expressing

Journal:

Article Title: CCL21 Expression Pattern of Human Secondary Lymphoid Organ Stroma Is Conserved in Inflammatory Lesions with Lymphoid Neogenesis

doi: 10.2353/ajpath.2007.061275

Figure Lengend Snippet: Distribution of periendothelial CCL21+ cells in HEVs of ectopic GC-like structures. Serial immunostaining for CD21 (A), CXCL13 (B), CCL21 (C), and PNAd (D) is shown on tissue sections of rheumatoid synovium, illustrating the presence of CCL21-expressing cells localized perivascularly (C) in close association to PNAd-positive HEVs (E) within a highly organized aggregate characterized by a CD21+ follicular DC network (A) and CXCL13 expression (D). In E and F, a detail of the vessel delimitated by the black square in C and E is shown at high magnification, revealing the presence of CCL21-expressing non-ECs (D) adherent to the abluminal side of the PNAd-positive HEV endothelium (F). Original magnifications: ×200 (A–D); ×1000 (D and F).

Article Snippet: After TBS washing and an additional blocking in 10% human serum diluted in TBS, goat anti-human CCL21 was applied, followed by incubations with donkey anti-goat IgG biotinylated (Jackson ImmunoResearch Laboratories) and rhodamine Red-X-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Immunostaining, Expressing

Journal:

Article Title: CCL21 Expression Pattern of Human Secondary Lymphoid Organ Stroma Is Conserved in Inflammatory Lesions with Lymphoid Neogenesis

doi: 10.2353/ajpath.2007.061275

Figure Lengend Snippet: Preserved relationship between CCL21 production and HEVs in human SLOs and ELTs. A–J: Immunostaining for CCL21 (A, C, E, G, and I; in red) and parallel immunostaining for PNAd (B, D, F, H, and J; in red) on paraffin-embedded tissues of human MLN (A and B), SS salivary gland (C and D), UC intestinal wall (E and F), CD intestinal wall (G and H), and RA synovium (I and J) reveal the presence of CCL21-expressing non-ECs adherent to the abluminal side of PNAd-positive HEVs both in human SLOs and within ectopic lymphoid aggregates in lymphoid neogenetic diseases. K and L: IHC (K) and in situ hybridization (L) for CCL21 in consecutive synovial tissue sections of RA confirm the complete co-localization between CCL21 protein expression (K, in red) and mRNA production (L, in black) within non-ECs surrounding a vessel with HEV morphology. Black arrows indicate blood vessel ECs lacking detectable levels of CCL21, despite the presence of adjacent CCL21-positive cells. M and N: Single color separations and color merges of double IF staining for CCL21 (in red) and PNAd (in green) on cryosections of human tonsil (M) and rheumatoid synovial tissue (N). The strongest CCL21 immunostaining is seen within PNAd-positive HEVs at their basal side, whereas PNAd staining is strongest at the luminal face. Original magnifications: ×1000 (A–J, M, and N); ×400 (K and L).

Article Snippet: After TBS washing and an additional blocking in 10% human serum diluted in TBS, goat anti-human CCL21 was applied, followed by incubations with donkey anti-goat IgG biotinylated (Jackson ImmunoResearch Laboratories) and rhodamine Red-X-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Immunostaining, Expressing, In Situ Hybridization, Staining

Journal:

Article Title: CCL21 Expression Pattern of Human Secondary Lymphoid Organ Stroma Is Conserved in Inflammatory Lesions with Lymphoid Neogenesis

doi: 10.2353/ajpath.2007.061275

Figure Lengend Snippet: SMA-positive stromal cells express CCL21 in human MLNs and in synovial ELT. Left: Stainings on paraffin-embedded sections of human MLN are shown. A and B: IHC for PNAd (A, in red) and double IHC for SMA (B, in brown) and CCL21 (B, in red) on serial tissue sections showing CCL21-positive cells in periendothelial localization around a PNAd-positive HEV and displaying SMA immunoreactivity. C–E: Single color separations (C and D) and double merge (E) of double IF for CCL21 (C and E; in red) and SMA (D and E; in green) show the co-localization between the two antibodies (revealed by yellow signal in E) in association to a vessel with HEV morphology. F: Double IHC for CCL21 (in red) and SMA (in brown) is shown on a tissue area at low magnification, demonstrating the co-localization between CCL21 and a network of SMA-positive cells distributed throughout the T area and connecting vascular structures. Note the absence of the SMA-positive reticulum inside a germinal center (black arrow). In G, a detail of the area represented in F is shown at high magnification. H–J: Single color separations (H and I) and color merge (J) of double IF for CCL21 (H and J; in red) and SMA (I and J; in green) show the co-localization between CCL21-expressing cells within the T-cell area and SMA-positive reticular cells, as revealed by yellow signal in J. Right: Stainings on paraffin-embedded sections of rheumatoid synovial membrane are shown. K and L: single IHC for PNAd (K, in red) and double IHC for SMA (L, in brown) and CCL21 (L, in red) on serial sections show that CCL21-positive cells adherent to a PNAd-positive HEV are SMA-positive. M–O: Single color separations (M and N) and color merge (O) of double IF for CCL21 (M and O; in red) and SMA (N and O; in green) confirms the co-localization of the two markers (yellow signal in O) in association to a synovial vessel with HEV morphology. P: Double IHC for CCL21 (in red) and SMA (in brown) is shown at low magnification on a synovial hypertrophic villous, demonstrating the co-localization between CCL21 and a network of SMA-positive cells connecting vascular structures within a lymphoid aggregate. In Q, high magnification of a detail of picture P is shown. R–T: Single color separations (R and S) and color merge (T) of double IF for CCL21 (R and T; in red) and SMA (S and T; in green) confirms the co-localization of the two markers (yellow signal in T) within reticular cells unrelated to vascular structures. Experiments analyzed under IF microscopy. Original magnifications: ×1000 (A–E, G–O, and Q–T); ×200 (F and P).

Article Snippet: After TBS washing and an additional blocking in 10% human serum diluted in TBS, goat anti-human CCL21 was applied, followed by incubations with donkey anti-goat IgG biotinylated (Jackson ImmunoResearch Laboratories) and rhodamine Red-X-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Microscopy

Journal:

Article Title: CCL21 Expression Pattern of Human Secondary Lymphoid Organ Stroma Is Conserved in Inflammatory Lesions with Lymphoid Neogenesis

doi: 10.2353/ajpath.2007.061275

Figure Lengend Snippet: Intracytoplasmic localization of CCL21 in SMA-positive cells. A–D: Double IF for CCL21 (in red) and SMA (in green) is shown in human MLN (A and B) and rheumatoid synovial membrane (C and D) analyzed by confocal microscopy. B and D show the images represented in A and B with DAPI counterstaining to highlight the cell nucleus (in blue). CCL21 signal is detected within SMA-positive reticular cells both in human SLOs and in the ELT of rheumatoid synovitis. E–H: Single color separations (E and F) and color merges (G and H) of double IF staining for CCL21 (E, G, H, in red) and SMA (F–H, in green) on a cytospin preparation obtained from freshly isolated LN cells, demonstrating co-localization of CCL21 and SMA ex vivo in two cells indicated by white arrows (yellow signal in G and H). In H, cell nuclei are counterstained in blue with DAPI. Arrowheads indicate a SMA-positive cell lacking CCL21 staining. I: CCL21 gene expression levels in intact LN, in the freshly isolated lymph node cells analyzed in E–H (LN FIC), and in a lymph node fibroblast cell line (LN CL) determined by quantitative PCR. RQ, relative quantification. Data are normalized for β-actin. Original magnifications, ×1000.

Article Snippet: After TBS washing and an additional blocking in 10% human serum diluted in TBS, goat anti-human CCL21 was applied, followed by incubations with donkey anti-goat IgG biotinylated (Jackson ImmunoResearch Laboratories) and rhodamine Red-X-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Confocal Microscopy, Staining, Isolation, Ex Vivo, Expressing, Real-time Polymerase Chain Reaction

Journal:

Article Title: CCL21 Expression Pattern of Human Secondary Lymphoid Organ Stroma Is Conserved in Inflammatory Lesions with Lymphoid Neogenesis

doi: 10.2353/ajpath.2007.061275

Figure Lengend Snippet: SMA-positive can express CCL19 and co-localize with mature DCs. A–D: Stainings on paraffin-embedded sections of human MLN are shown. A: Double IHC for CCL19 (in red) and SMA (in brown) is shown on a tissue area at high magnification, demonstrating the co-localization between CCL19 and a network of spindle-shaped SMA-positive cells distributed throughout the T area. B–D: Color merge (B) and single color separations (C and D) of double IF for CCL19 (B and C; in red) and SMA (B and D; in green) confirm the co-localization between CCL19-expressing cells within the T-cell area and SMA-positive reticular cells, as revealed by yellow signal in B. E–H: Stainings on paraffin-embedded sections of rheumatoid synovial membrane are shown. E: Double IHC for CCL19 (in red) and SMA (in brown) reveals co-localization between CCL19 and SMA within perivascular cells in a lymphoid aggregate, indicated by black arrows. Some scattered CCL19-expressing cells are SMA-negative (arrowheads). F–H: Color merge (F) and single color separations (G and H) of double IF for CCL19 (F and G; in red) and SMA (F and H; in green) confirm the co-localization between CCL19-expressing and SMA-positive cells, as revealed by yellow signal in F. Note again CCL19-positive SMA-negative cells (arrowheads). I–K: Double IHC for CCL21 (in red) and the mature DC marker DC-LAMP (in brown) in rheumatoid synovium sections. I: In lymphoid aggregates, perivascular CCL21-expressing cells show area co-localization with DC-LAMP-positive DCs. DC-LAMP-positive DCs are also found in proximity (J) and within (K) CCL21-positive lymphatic vessels in synovial areas of cell infiltration. Original magnifications, ×1000.

Article Snippet: After TBS washing and an additional blocking in 10% human serum diluted in TBS, goat anti-human CCL21 was applied, followed by incubations with donkey anti-goat IgG biotinylated (Jackson ImmunoResearch Laboratories) and rhodamine Red-X-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Marker

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Suppression of plasmacytoid dendritic cell migration to colonic isolated lymphoid follicles abrogates the development of colitis.

doi: 10.1016/j.biopha.2021.111881

Figure Lengend Snippet: Fig. 4. Effects of 1 μM As-IV or Oxy on RAC1 activation. RAC1 activation was detected by evaluating the expression of the GTP-RAC1 protein by using a western blot analysis kit. A representative image and the quantification of the band intensity for GTP-RAC1 relative to that of total RAC1 are shown in A-C. (A) Representative western blot analysis of GTP-RAC1 in BMpDCs induced with CCL21 and quantification of the band intensity are shown (5 independent experiments). Data are expressed as the mean ± SE (*p < 0.05, n = 5). Representative western blot analysis of GTP-RAC1 in CCL21-induced migrated BMpDCs following treatment with As- IV (B) or Oxy (C) and the quantification of band intensity are shown (7 independent experiments). Data are expressed as the mean ± SE (*p < 0.05, n = 7). Microarray analysis of BMpDCs induced with a CCL21 gradient following treatment with As-IV or Oxy was performed by using the Clariom S Array Mouse. The differential expression of genes in BMpDCs induced with a CCL21 gradient following treatment with vehicle (control), As-IV or Oxy is displayed in the heatmap (D). The red and blue colors indicated the up-regulation normalized intensity values (log2) and down-regulation normalized intensity values (log2) of each RNA in each sample.

Article Snippet: 30 μm sections cut by using a cryostat (Leica, Nussloch, Germany) were soaked in 0.3% Triton X (Sigma, Missouri, USA) for 2 h and 2% Block Ace (DS Pharma Biomedical, Osaka, Y. Zhang et al. Biomedicine & Pharmacotherapy 141 (2021) 111881 Japan) for 1 h. Then, the colon sections were stained with the primary antibodies rat IgG anti-mouse B220 (1:200, BioLegend, San Diego, CA, USA), hamster IgG anti-mouse CD11c (1:100, BioLegend) and goat IgG anti-mouse CCL21 (1:200, R&D Systems).

Techniques: Activation Assay, Expressing, Western Blot, Microarray, Quantitative Proteomics, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Suppression of plasmacytoid dendritic cell migration to colonic isolated lymphoid follicles abrogates the development of colitis.

doi: 10.1016/j.biopha.2021.111881

Figure Lengend Snippet: Fig. 6. Effects of As-IV or Oxy on the distribution of CCL21 in the DSS-induced colitis model. The distribution of CCL21 in the colonic ILFs of DSS-induced colitis mice treated with saline (A), As-IV (B) or Oxy (C) was identified by immunohistochemical staining of the colon. Immunohistochemical staining was performed on samples from 3 mice/group, and representative images are presented.

Article Snippet: 30 μm sections cut by using a cryostat (Leica, Nussloch, Germany) were soaked in 0.3% Triton X (Sigma, Missouri, USA) for 2 h and 2% Block Ace (DS Pharma Biomedical, Osaka, Y. Zhang et al. Biomedicine & Pharmacotherapy 141 (2021) 111881 Japan) for 1 h. Then, the colon sections were stained with the primary antibodies rat IgG anti-mouse B220 (1:200, BioLegend, San Diego, CA, USA), hamster IgG anti-mouse CD11c (1:100, BioLegend) and goat IgG anti-mouse CCL21 (1:200, R&D Systems).

Techniques: Saline, Immunohistochemical staining, Staining