Journal: bioRxiv

Article Title: Single-cell transcriptional landscape of temporal neutrophil response to burn wound in larval zebrafish

doi: 10.1101/2024.04.01.587641

Figure Lengend Snippet: (A) Schematic view of validating signatures identified from zebrafish myeloid subsets in human burn patient expression profile. (B) Expression level distribution of Immature_Neuts signatures (top panel) and lgals3bpb_Mac1 (bottom panel) by patient TBSA shown as raincloud plots. Paired T-tests were performed between the Low and the Very High TBSA groups. (C) Expression distribution of GPR84 and GYG1 in patients by TBSA level. Spearman correlation (ρ) is calculated between gene expression and TBSA level. (D) Representative flow plots (left) and quantifications of frequencies shown as percentages (right) of GPR84+ neutrophils in burn (n = 6) and healthy (n = 4) blood. Paired samples t-test, p = 0.022.

Article Snippet: Protein-targeted staining was done with the following antibodies in Cell Staining Buffer: Alexa Fluor 700-conjugated anti-human CD16 at 1:400, PE/Fire-640-conjugated anti-human CD66b at 1:400, PerCP-conjugated anti-human CD3 at 1:200, PerCP-conjugated anti-human CD19 at 1:200, PerCP-conjugated anti-human CD56 at 1:400, PerCP-conjugated anti-human CD203c at 1:100 (BioLegend), BUV395-conjugated mouse anti-human CD45 at 1:100 (BD Biosciences), and PE-conjugated GPR84 at 1:200 (Alomone Labs).

Techniques: Expressing, Gene Expression

Journal: Bioengineering

Article Title: RNU ( Foxn1 RNU -Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats

doi: 10.3390/bioengineering8010012

Figure Lengend Snippet: CD68+ staining is increased in RNU DMM-treated sites compared to Sprague Dawley. CD68 staining was quantified and showed increased levels in RNU rats ( A ). Yet, CD163 ( B ) and FoxP3 ( C ) were unaltered. CD4 staining was increased in RNU rats ( D ) while CD8 staining was lower ( E ). In addition, CD4, CD8, and FoxP3 levels were low. DRAQ5 showed no change in DNA staining ( F ). Data shown are means ± SEM of 8 animals. Letters not shared indicated a significant difference ( p < 0.05, Tukey).

Article Snippet: Primary antibodies used in this experiment were: mouse anti-Pax7 (ab55494, Abcam, Cambridge, UK); rabbit anti-nicotinic acetylcholine receptor-epsilon (AChR-ε, ab65180, Abcam); mouse anti-nicotinic acetylcholine receptor-gamma (AChR-γ, MA3-043, Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-myosin heavy chain-fetal (fMyHC, SC-53097, Santa Cruz Biotechnology); CD68 (ab125212, Abcam); CD163 (ab87099, Abcam); CD8 (MAB116, R&D Systems), CD4 (MAB554, R&D Systems), FoxP3 (MAB8214, R&D Systems) and were diluted in PBS with 1% BSA and 0.3% Tween-20.

Techniques: Staining

Journal: Cancer Science

Article Title: Angiotensin II subtype 1a receptor signaling in resident hepatic macrophages induces liver metastasis formation

doi: 10.1111/cas.13306

Figure Lengend Snippet: Angiotensin II (Ang II )/ AT 1a axis enhances transforming growth factor‐β1 ( TGF ‐β1) expression in Kupffer cells. (a) Accumulation of F4/80 + cells, TGF ‐β1 + cells, and F4/80 + / TGF ‐β1 + cells in metastatic areas from WT and AT 1a KO mice on day 14. Double‐staining of liver sections with antibodies against F4/80 (red) and TGF ‐β1 (green) in WT and AT 1a KO mice. Expression of TGF ‐β1 was colocalized with F4/80 + cells. Metastatic area is delineated with the white dashed line. T, tumor. Scale bar = 100 μm. (b–d) Numbers of F4/80 + cells (b), TGF ‐β1 + cells (c), and F4/80 + TGF ‐β1 + cells (d) in metastatic areas from WT and AT 1a KO mice. Data are expressed as the means ± SD of six mice per group. * P < 0.05. (e) Expression of AT 1a and AT 1b in KUP 5 Kupffer cells. Data are expressed as the means ± SD of six mice per group. (f) Expression of TGF ‐β1 on KUP 5 cells under stimulation with Ang II . Expression of TGF ‐β1 was enhanced 6 h after stimulation with Ang II compared with control. There was no significant difference at 12 h. Data are expressed as the means ± SD of six mice per group. * P < 0.05 versus control.

Article Snippet: For the latter, sections were activated using Histo VT One (Nacalai Tesque, Yokohama, Japan) and then incubated overnight at 4°C with one of the following primary antibodies: (a) anti‐mouse F4/80 antibody (1:200, rat monoclonal, sc52664; Santa Cruz Biotechnology, Dallas, TX, USA); (b) anti‐mouse TGF‐β1 antibody (1:200, rabbit polyclonal, ab92486; Abcam, Cambridge, UK); (c) anti‐mouse GFP antibody (1:200, rabbit polyclonal, ab290; Abcam); (d) anti‐mouse Ang II type 1A receptor antibody (1:100, rabbit polyclonal, bs‐2132R; Bioss, Boston, MA, USA); (e) anti‐mouse desmin antibody (1:100, goat polyclonal, ab80503; Abcam); (f) anti‐mouse type I collagen antibody (1:100, rabbit polyclonal, ab21286; Abcam); or (g) anti‐mouse CD31 antibody (1:200, rabbit polyclonal, ab28364; Abcam).

Techniques: Expressing, Double Staining

Journal: Cancers

Article Title: Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer

doi: 10.3390/cancers12061626

Figure Lengend Snippet: Validation of ex vivo BMRC/CTC using complementary imaging platforms. ( A ) Detection of ex vivo human CTCs by CellSearch CTC (epithelial) kit. The kit enumerates CTCs using DAPI, Pan-cytokeratin-PE (CK-PE), and CD45-APC staining. Upper panel shows a CTC clustering with a CD45+ cell (lymphocyte); lower panels show an individual CTC. Image resolution achieved in accordance with the USFDA-approved CellSearch platform. ( B ) Immunofluorescent (IF) staining of ex vivo BMRC using GCDFP15/MG cocktail. Image of a BMRC showing red IF staining against mouse primary antibody clones 23A3 (GCDFP-15) and 304-1A5 (MG), and green IF staining for rabbit clone 31A5 (MG). ( C ) IF images of single ex vivo epithelial and stem-like BMRCs and CTCs. ( D ) DEPArray images of ex vivo epithelial and stem-like BMRCs and CTCs. Image magnification: 10×. The DEPArray platform allows the capture, visualization, and downstream interrogation of single CTCs. Since the image resolution and magnification obtained from FDA-cleared CellSearch and DEPArray platforms cannot be altered by the user, we used IF to provide a high-resolution image of captured ex vivo BMRC/CTCs.

Article Snippet: IHC on mouse tissue using antibodies of mouse origin were performed using M.O.M. elite peroxidase kit; dual IHCs were performed using ImmPRESS Duet Double Staining HRP/AP Polymer Kit, and triple IHCs were performed by multiplexing with ImmPRESS-AP Anti-Rat IgG, Mouse Adsorbed Polymer Detection Kit (Vector Labs, Burlingame, CA, USA).

Techniques: Ex Vivo, Imaging, Staining, Clone Assay

Journal:

Article Title: Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

doi: 10.1128/JVI.77.18.9769-9779.2003

Figure Lengend Snippet: Syncytium-inducing activity of p10 constructs. Cells were transfected with various p10 constructs and fixed at 36 h posttransfection. The fixed cells were immunostained with anti-HA monoclonal antibodies and goat anti-mouse IgG conjugated with alkaline phosphatase. Arrows in the left panels indicate the boundaries of antigen-positive syncytial foci. The brighter areas surrounded by darkly stained areas indicate antigen-positive cytoplasm surrounding multiple antigen-negative nuclei in a single syncytium. Arrows in the right panels indicate single, darkly staining antigen-positive cells in monolayers transfected with syncytium-negative p10 constructs. Magnification, ×100. A subset of p10 constructs are shown, which include substitutions that did not significantly alter the fusogenic activity of p10 (K67R), those that drastically reduced both the number and size of syncytia (G48A), and those that eliminated p10-induced syncytium formation (C63A, K67 M, and G50A). The qualitative fusion activities of all of the p10 constructs, assessed in a similar manner, are summarized in Fig. ​Fig.1C1C.

Article Snippet: Following extensive cold washes with HBSS, cells were fixed with ice-cold methanol for 2 min and incubated for 45 min with fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G (IgG) (Life Technologies) diluted 1:25 in phosphate-buffered saline (PBS)-bovine serum albumin.

Techniques: Activity Assay, Construct, Transfection, Staining

Journal:

Article Title: Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

doi: 10.1128/JVI.77.18.9769-9779.2003

Figure Lengend Snippet: Surface localization of p10 constructs. Live cells transfected with various p10 constructs were treated with anti-HA monoclonal antibodies to detect the presence of the surface-localized N-terminal HA epitope tags on the p10 constructs. Surface-bound anti-HA antibodies were detected by immunofluorescence with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. A subset of the p10 constructs are shown. All of the p10 constructs, assessed in the same manner, yielded similar results. Images were captured under identical parameters. Magnification, ×630; bars, 20 μm. The specificity of the surface-staining protocol was confirmed by using a p10 construct with HA tags in the C-terminal endodomain, as previously described (45), and is evident from the low level of background staining of the surrounding nontransfected cells in the confluent monolayer.

Article Snippet: Following extensive cold washes with HBSS, cells were fixed with ice-cold methanol for 2 min and incubated for 45 min with fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G (IgG) (Life Technologies) diluted 1:25 in phosphate-buffered saline (PBS)-bovine serum albumin.

Techniques: Construct, Transfection, Immunofluorescence, Staining

Journal: Journal of translational medicine

Article Title: Niclosamide suppresses the expansion of follicular helper T cells and alleviates disease severity in two murine models of lupus via STAT3.

doi: 10.1186/s12967-021-02760-2

Figure Lengend Snippet: Fig. 1 Niclosamide ameliorates disease aggravation in MRL/lpr mice. Female 10-week-old MRL/lpr mice were orally administered 0.5% methyl cellulose (vehicle, n = 7), or 100 mg/kg niclosamide (Niclosamide, n = 7) daily until they were 16-week-old. a Left, Representative photographs documenting the enlargement of kidneys. Right, kidney weights in vehicle, niclosamide groups. b Urine albumin levels normalized to creatinine. c Serum levels of anti-dsDNA IgG antibody. d Serum levels of antibody subclasses (IgG, IgG1, and IgM). e Serum levels of IL-6 and IL-21. f Left, representative photomicrographs of PAS-stained sections of kidney. Original magnification ×200 (upper), ×100 (middle, bottom). Right, histological score. g Left, representative immunofluorescent images of kidney C3 staining. Original magnification ×100 (upper), ×400 (bottom). Right, MFI of C3 deposition. Data shown as mean ± SD. t-test was performed. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Total IgG, IgG1, IgG2a, and IgM levels in the sera of the mice were measured by ELISA following the manufacturer’s instructions (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Staining

Journal: Journal of translational medicine

Article Title: Niclosamide suppresses the expansion of follicular helper T cells and alleviates disease severity in two murine models of lupus via STAT3.

doi: 10.1186/s12967-021-02760-2

Figure Lengend Snippet: Fig. 4 Niclosamide inhibits TFH-like cell differentiation and B cell IgG production in vitro. Naive CD4+ T cells were purified from the spleens of MRL/lpr and C57BL/6 mice. For TFH-like cell differentiation, purified CD4+ T cells were activated with mouse T-activator CD3/CD28 Dynabeads, and treated with 20 ng/ml IL-6, 20 ng/ml IL-21, 10 μg/ml anti-IL-4, 10 μg/ml anti-IFN-γ, and 20 μg/ml anti-TGF-β for 4 days with or without niclosamide. a Left, TFH-like cells (CXCR5+PD-1+, gated on CD4+) isolated from MRL/lpr mice were analyzed by flow cytometry. Right, the percentage of TFH-like cells is shown. b p-STAT3 and TCF-1 expressions in TFH-like cells isolated from MRL/lpr mice were analyzed by western blot. c mRNA expression levels of Bcl-6, CXCR5, and Blimp-1 in TFH-like cells isolated from MRL/lpr mice were measured by real-time PCR. d TFH-like cells and B cells isolated from C57BL/6 mice were co-cultured with or without TFH-like cells for 3 days, and then the concentrations of IgG in the supernatants were detected by ELISA. Data shown as mean ± SD. One-way ANOVA was performed. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Article Snippet: Total IgG, IgG1, IgG2a, and IgM levels in the sera of the mice were measured by ELISA following the manufacturer’s instructions (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Cell Differentiation, In Vitro, Purification, Isolation, Flow Cytometry, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of translational medicine

Article Title: Niclosamide suppresses the expansion of follicular helper T cells and alleviates disease severity in two murine models of lupus via STAT3.

doi: 10.1186/s12967-021-02760-2

Figure Lengend Snippet: Fig. 5 Niclosamide ameliorates disease aggravation in R848-induced mice. Female 8-week-old C57BL/6 mice were treated with 50 μg of the TLR7 agonist R848 or control (acetone) three times weekly and were orally administered 0.5% methyl cellulose (control, R848, n = 4–6), or 100 mg/kg niclosamide (niclosamide, n = 7) daily until they were 12-week-old. a Representative photographs documenting the enlargement of spleens and cLNs. b Spleen lengths and weights in each group. c cLN lengths and weights in each group. d Urine albumin levels normalized to creatinine. e Serum levels of anti-dsDNA IgG antibody. f Serum levels of antibody subclasses (IgG, IgG2a). g Serum levels of IL-6, IL-21. h Left, representative photomicrographs of PAS-stained sections of kidney. Original magnification ×200 (upper), ×100 (bottom). Right, histological scores. i Left, representative immunofluorescent images of kidney C3 staining. Original magnification ×100 (upper), ×400 (bottom). Right, MFI of C3 deposition. Data shown as mean ± SD. One-way ANOVA was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Article Snippet: Total IgG, IgG1, IgG2a, and IgM levels in the sera of the mice were measured by ELISA following the manufacturer’s instructions (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Control, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activated protein C attenuates systemic lupus erythematosus and lupus nephritis in MRL-Fas(lpr) mice.

doi: 10.4049/jimmunol.1101125

Figure Lengend Snippet: FIGURE 1. Recombinant activated protein C sup- presses renal pathology in MRL-Fas(lpr) mice. A, Renal sections of 18-wk-old MRL-Fas(lpr) mice were stained with PAS. Representative PAS staining kidney sections showed glomerular damage in vehi- cle-injected mice when compared with activated pro- tein C-injected mice. MRL control mice showed no glomerular abnormalities. Images are representative for 15 mice in each treatment group and 5 MRL wt mice. Renal sections of 18-wk-old MRL-Fas(lpr) mice were stained with Abs for IgG and comple- ment C3c as indicated. Note that activated protein C-injected mice show less glomerular IgG and C3c deposits. Images are representative of 15 mice in each group. Original magnification 3400 (PAS and IgG) or 3200 (C3c). B, Quantitative measures of lupus nephritis in MRL-Fas(lpr) mice. The lupus nephritis disease activity index (score ranging from 0–24) and the lupus nephritis chronicity index (score ranging from 0–12) were determined as markers of kidney damage in lupus nephritis. Glomerular IgG deposition was compared between vehicle- and acti- vated protein C-treated groups (score ranging from 0–3). Data are expressed as means 6 SEM. *p , 0.05, **p , 0.01 versus vehicle group (vehicle, n = 15; activated protein C [APC], n = 15).

Article Snippet: The assay was developed with HRP-labeled goat anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3, and IgM Abs (Bethyl Laboratories).

Techniques: Recombinant, Staining, Injection, Control, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activated protein C attenuates systemic lupus erythematosus and lupus nephritis in MRL-Fas(lpr) mice.

doi: 10.4049/jimmunol.1101125

Figure Lengend Snippet: FIGURE 4. Activated protein C reduces autoantibodies in MRL-Fas(lpr) mice. Plasma levels of total IgG (A) and total IgM (B) were determined by ELISA. C, ANA staining patterns on Hep2 human epithelial cells for serum, derived from MRL wt, vehicle-treated, and activated protein C- treated MRL-Fas(lpr) mice at 1:200 dilution. Original magnification 31000. Plasma levels of total IgG against dsDNA (D) or rheumatoid factor (E) were determined by ELISA. Data are expressed as means 6 SEM (n = 15 each treament group; n = 5 MRL/wt). *p , 0.05, **p , 0.01 versus vehicle group.

Article Snippet: The assay was developed with HRP-labeled goat anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3, and IgM Abs (Bethyl Laboratories).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay