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: 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

Journal: bioRxiv

Article Title: Microglial and Astrocyte priming in the APP/PS1 model of Alzheimer’s Disease: increased vulnerability to acute inflammation and cognitive deficits

doi: 10.1101/344218

Figure Lengend Snippet: (A-C) Chemokine secretion in microglia and astrocyte supernatants, 6h after IL-1β challenge (2.5 ng/mL) measured by ELISA for CCL2, CXCL1 and CXCL10. Two way ANOVA, *p<0.001, compared to astrocyte control; #p<0.001, compared to microglia IL-1β. (D) Light microscopy labelling of CCL2 in WT and Tg mice, 2h after i.c. challenge with IL-1β (10 ng) or saline. (E) Confocal imaging of Tg+IL-1β group for CCL2 (red; 594nm) and GFAP (green; 488nm). Iba-1 (green; 488nm) is captured in the inset. (F) Light microscopy labelling of CXCL1. (G) Light microscopy labelling of CXCL10. Insets show detail of positive vascular and astrocytic cells for the different chemokines.

Article Snippet: For confocal microscopy, sections were labelled with the following combinations: 6E10 (1:1000; Biolegend 803001) with an Alexa Fluor 488 anti-mouse (1:800; Invitrogen) together with Iba-1 (1:2000; Abcam ab5076) Alexa Fluor 594 anti-goat (1:800; Invitrogen); 6E10 with an Alexa Fluor 633 anti-mouse (1:800; Invitrogen) in combination with IL-1β (1:50; Peprotech 500-P51) with an Alexa Fluor 488 anti-rabbit (1:800; Invitrogen); 6E10 with an Alexa Fluor 633 anti-mouse and GFAP with an Alexa Fluor 488 anti-rabbit; GFAP (1:2000; Dako Z0334) with an Alexa Fluor 594 anti-rabbit (1:800; Invitrogen) together with IL-1β (1:50; Peprotech 500-P51) with an Alexa Fluor 488 anti-rabbit; GFAP with an Alexa Fluor 488 anti-rabbit in combination with CCL2 (1:200; R&D AF-479-NA) with Alexa Fluor 594 anti-goat; Iba-1 (1:2000; Abcam ab5076) with Alexa Fluor 594 anti-goat.

Techniques: Enzyme-linked Immunosorbent Assay, Light Microscopy, Imaging

Journal: bioRxiv

Article Title: Microglial and Astrocyte priming in the APP/PS1 model of Alzheimer’s Disease: increased vulnerability to acute inflammation and cognitive deficits

doi: 10.1101/344218

Figure Lengend Snippet: Exogenous i.c. administration of LPS in normal mice induces activation of microglial cells to produce regulated secretion of IL-1β and this IL-1β may activate astrocytes to release chemokines such as CCL2, CXCL1 or CXCL10. However, in APP/PS1 mice, LPS stimulates more robust production and processing of IL-1β from microglia primed by Amyloid pathology. Astrocytes primed by amyloid pathology also respond in an exaggerated way to exogenous IL-1β, producing enhanced levels of chemokines. If levels of IL-1, arising endogenously from microglial stimulation, are already exaggerated this will produce a further amplification upon stimulating astrocytes that are hypersensitive to IL-1. These exaggerated responses also appear to affect many other astrocytic transcripts involved in innate immune function as shown by fold-change expression in comparison to WT+saline.

Article Snippet: For confocal microscopy, sections were labelled with the following combinations: 6E10 (1:1000; Biolegend 803001) with an Alexa Fluor 488 anti-mouse (1:800; Invitrogen) together with Iba-1 (1:2000; Abcam ab5076) Alexa Fluor 594 anti-goat (1:800; Invitrogen); 6E10 with an Alexa Fluor 633 anti-mouse (1:800; Invitrogen) in combination with IL-1β (1:50; Peprotech 500-P51) with an Alexa Fluor 488 anti-rabbit (1:800; Invitrogen); 6E10 with an Alexa Fluor 633 anti-mouse and GFAP with an Alexa Fluor 488 anti-rabbit; GFAP (1:2000; Dako Z0334) with an Alexa Fluor 594 anti-rabbit (1:800; Invitrogen) together with IL-1β (1:50; Peprotech 500-P51) with an Alexa Fluor 488 anti-rabbit; GFAP with an Alexa Fluor 488 anti-rabbit in combination with CCL2 (1:200; R&D AF-479-NA) with Alexa Fluor 594 anti-goat; Iba-1 (1:2000; Abcam ab5076) with Alexa Fluor 594 anti-goat.

Techniques: Activation Assay, Amplification, Expressing