Journal: iScience

Article Title: Host-Microbiome Interactions in Distinct Subsets of Preterm Labor and Birth

doi: 10.1016/j.isci.2023.108341

Figure Lengend Snippet: Figure 5. Local immune responses in the cervix with signs of early premature labor or active premature labor (A) Dams were intra-amniotically injected under ultrasound guidance with PBS, LPS, or IL-1a, or subcutaneously with DMSO or RU-486 (n = 3 dams per group). The cervix was collected with signs of early premature labor (SEL; 6 h) and histology was utilized to evaluate structure by Movat pentachrome staining, leukocyte infiltration by immunohistochemistry, and leukocyte subsets by multiplex immunofluorescence. (B) Representative Movat pentachrome staining images of the cervix from control (PBS and DMSO) and preterm labor (LPS, IL-1a, and RU-486) mice with SEL. Blue staining indicates mucin, yellow indicates collagen, and red/bright red indicates muscle/fibrin. Nuclei appear as dark blue/black. (C) Representative images showing 3,30diaminobenzidine (DAB) immunohistochemistry to detect the pan-leukocyte marker CD45 in the cervix of control (PBS and DMSO) and premature labor (LPS, IL-1a, and RU-486) mice (n = 3 per group) with SEL. (D‒F) Semi-quantification of the percentage of CD45+ cells among all cells for each study group comparison. Data are shown as dot plots where midlines indicate medians and whiskers indicate minimum and maximum values. p values for the comparisons between groups were determined by unpaired t tests. *p < 0.05. (G‒I) Representative merged images showing the co-localized immunofluorescence detection of neutrophils (Ly6G+ cells, pink), monocytes/macrophages (F4/ 80+ cells, green), T cells (CD3+ cells, red), B cells (CD19+ cells, light pink), and NK cells (NCR1+ cells, yellow) in the cervices of treated mice (LPS, IL-1a, and RU-486) (n = 3 per group) with SEL. Nuclear staining is shown in blue (40,6-diamidino-2-phenylindole; DAPI). Images were taken at 20X magnification. Scale bars represent 50mm. See also Figure S6.

Article Snippet: Following optimization, the detection panel was generated in the following order (antibody followed by OPAL fluorophore): NCR1 (polyclonal rabbit anti-mouse antibody; cat. Ab214468, Abcam, Cambridge, England) with OPAL 520, F4/80 (monoclonal rabbit anti-mouse antibody; clone D2S9R; cat. 24 iScience 26, 108341, December 15, 2023 70076S, Cell Signaling Technology, MA, USA) with OPAL 540, CD3e (monoclonal rabbit anti-mouse antibody; clone E4T1B; cat. 78588S, Cell Signaling Technology) with OPAL 570, CD19 (monoclonal rabbit anti-mouse antibody; clone: D4V4B; cat. 90176S, Cell Signaling Technology) withOPAL 620, and Ly6G (monoclonal rabbit anti-mouse antibody; clone E6Z1T; cat. 87048S, Cell Signaling Technology) withOPAL 690.

Techniques: Injection, Staining, Immunohistochemistry, Multiplex Assay, Control, Marker, Comparison

Journal: Arthritis Research & Therapy

Article Title: IL-35 enhances IL-10⁺ breg-mediated immunoregulation and attenuates inflammation and fibrosis in systemic sclerosis

doi: 10.1186/s13075-026-03735-8

Figure Lengend Snippet: Expression levels of B cell subsets and related cytokines in peripheral blood of systemic sclerosis patients and healthy controls. A Flow cytometry gating strategy and staining patterns showing frequencies of B cells, interleukin-10+ regulatory B cells (IL-10+Breg), IL-35+Breg cells, and IL-6+ effector B cells (IL-6+Beff). B Plasma concentrations of IL-35, IL-10, B cell activating factor (BAFF), and IL-6. C Relative mRNA expression of IL-10, IL-6, and transforming growth factor-β1 (TGF-β1) in skin tissue. D Immunofluorescence images of IL-10+Breg cells in skin tissue and their proportion among CD19+B cells (scale bar: 50 μm, 400× magnification). E Immunofluorescence images of IL-35+Breg cells in skin tissue and their proportion among CD19+B cells (scale bar: 50 μm, 400× magnification). F Heatmap showing correlations between circulating B cell subsets/cytokines and clinical parameters. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant

Article Snippet: The following reagents were used: Rat polyclonal CD19 antibody (Columbia, MD, USA; Cat# 32727), a Mouse IL-6 antibody (Santa Cruz Biotechnology, USA; Cat# SC-28343), Mouse monoclonal CD19 antibody (Novus Biologicals, USA; Cat# NBP2-25196SS), Rat Anti-IL-10 antibody (Abcam, Cambridge, UK; Cat# ab133575), and Rat IL-35 Antibody (Abbexa, Cambridge, UK; Cat# Abx274658),

Techniques: Expressing, Flow Cytometry, Staining, Clinical Proteomics, Immunofluorescence