rabbit anti mouse/rat urokinase (upa) polyclonal fractionated,rabbit anti-mouse urokinase igg fraction  (Innovative Research Inc)

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Expression of fibrinolytic system components in 24 h and 7 days old venous thrombi. Panels A-F show the expression (red) of (A-B) tPA, (C-D) uPA, (E-F) PAI-1, (G-H) plasminogen (Pg), (I-J) α2AP, and (K-L) their quantification in 8-µm cryosections of 24 hours and 7 days thrombi as labeled. Red color (Alexa Fluor 555) represents each component as labeled and blue color shows 4′,6-diamidino-2-phenylindole–stained nuclei. For comparison, IVC from sham mice were immunostained and quantified for each antibody under the same color range in the histogram mode of Image Pro-Plus software. The total immunostained area (arbitrary units; AU/mm2) for each protein was measured in 500 μm (original magnification ×4) images of an IVC sample. (N = 4-5 per group, mean ± SEM). *P < .05, **P < .01, ***P < .001.

Article Snippet: The primary antibodies include rabbit anti-mouse against tPA (ASMTPA-GF; Molecular Innovations, Novi, MI), uPA (ASMUPA-GF-HT; Molecular Innovations, Novi, MI), PAI-1 (IASMPAI-GF; Innovative Research, Novi, MI), plasminogen (Abcam; Cambridge, MA); goat anti-mouse α2AP (AF1239; R&D Systems, Minneapolis, MN); rat anti-mouse Ly6G (clone1A8, #127602; BioLegend, San Diego, CA), and Ly6B.2 (#771G; Bio-Rad Laboratories, Hercules, CA).

Techniques: Expressing, Labeling, Staining, Software

Journal: Frontiers in Immunology

Article Title: Crucial Involvement of IL-6 in Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes

doi: 10.3389/fimmu.2019.03150

Figure Lengend Snippet: Sequences of the primers used for real-time RT-PCR.

Article Snippet: The following mAbs and polyclonal Abs (pAbs) were used for immunohistochemical and double-color immunofluorescence analyses: rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical Company, Japan), mouse anti-IL-6 mAb (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse IL-6 pAbs, rabbit anti-mouse CCL2 pAbs (Novus Biologicals, Centennial, CO), goat anti-mouse MMP-2 pAbs, goat anti-mouse MMP-9 pAbs (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse PLAU (uPA) pAbs, rabbit anti-mouse tPA pAbs, rabbit anti-mouse PAI-1 pAbs (Santa Cruz Biotechnology, Dallas, TX), mouse anti-mouse Col1A2 mAb (Santa Cruz Biotechnology), rabbit anti-mouse myeloperoxidase (MPO) pAbs (Neomarkers, Fremont, CA), rabbit anti-mouse CD3 mAb (Abcam, Tokyo, Japan), cyanine dye 3-conjugated donkey anti-rat IgG pAbs, cyanine dye 3-conjugated donkey anti-goat IgG, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG pAbs, and FITC-conjugated donkey anti-rabbit IgG pAbs (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Crucial Involvement of IL-6 in Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes

doi: 10.3389/fimmu.2019.03150

Figure Lengend Snippet: Intrathrombotic expression of IL-6 in wild-type (WT) mice after inferior vena cava (IVC) ligation. (A) Il6 gene expression was examined by real-time reverse transcription (RT)–PCR as described in section Materials and Methods. All values represent mean ± SEM ( n = 6). (B) Intrathrombotic IL-6 protein levels were determined by ELISA. All values represent mean ± SEM ( n = 6). (C) Immunohistochemical analysis of intrathrombotic IL-6 expression (original magnification, ×100, upper panel; ×400, lower panel). (D) A double-color immunofluorescence analysis of IL-6-expressing cells in the thrombus. The samples were immunostained with the combination of anti-F4/80 mAb and anti-IL-6 pAbs as described in section Materials and Methods. The fluorescent images were digitally merged in the right panel. Representative results from six independent experiments are shown here [original magnification, ×400; blue, nuclear staining by 4′,6-diamidino-2-phenylindole (DAPI)].

Article Snippet: The following mAbs and polyclonal Abs (pAbs) were used for immunohistochemical and double-color immunofluorescence analyses: rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical Company, Japan), mouse anti-IL-6 mAb (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse IL-6 pAbs, rabbit anti-mouse CCL2 pAbs (Novus Biologicals, Centennial, CO), goat anti-mouse MMP-2 pAbs, goat anti-mouse MMP-9 pAbs (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse PLAU (uPA) pAbs, rabbit anti-mouse tPA pAbs, rabbit anti-mouse PAI-1 pAbs (Santa Cruz Biotechnology, Dallas, TX), mouse anti-mouse Col1A2 mAb (Santa Cruz Biotechnology), rabbit anti-mouse myeloperoxidase (MPO) pAbs (Neomarkers, Fremont, CA), rabbit anti-mouse CD3 mAb (Abcam, Tokyo, Japan), cyanine dye 3-conjugated donkey anti-rat IgG pAbs, cyanine dye 3-conjugated donkey anti-goat IgG, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG pAbs, and FITC-conjugated donkey anti-rabbit IgG pAbs (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Ligation, Gene Expression, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Crucial Involvement of IL-6 in Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes

doi: 10.3389/fimmu.2019.03150

Figure Lengend Snippet: Intrathrombotic expression of proteolytic enzymes in wild-type (WT) and Il6 −/− mice. (A–C) A double-color immunofluorescence analysis of MMP-2-, MMP-9-, or PLAU-expressing cells in the thrombus. The fluorescent images were digitally merged in the right panel. Representative results from six independent experiments are shown here [original magnification, ×400; blue, nuclear staining by 4′,6-diamidino-2-phenylindole (DAPI)]. (D–F) Intrathrombotic gene expression of Mmp2 (D) , Mmp9 (E) , and Plau (F) after inferior vena cava (IVC) ligation. Each gene expression was determined by real-time reverse transcription (RT)–PCR as described in section Materials and Methods. All values represent the mean ± SEM ( n = 6 animals). * p < 0.05, ** p < 0.01, WT vs. Il6 −/− .

Article Snippet: The following mAbs and polyclonal Abs (pAbs) were used for immunohistochemical and double-color immunofluorescence analyses: rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical Company, Japan), mouse anti-IL-6 mAb (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse IL-6 pAbs, rabbit anti-mouse CCL2 pAbs (Novus Biologicals, Centennial, CO), goat anti-mouse MMP-2 pAbs, goat anti-mouse MMP-9 pAbs (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse PLAU (uPA) pAbs, rabbit anti-mouse tPA pAbs, rabbit anti-mouse PAI-1 pAbs (Santa Cruz Biotechnology, Dallas, TX), mouse anti-mouse Col1A2 mAb (Santa Cruz Biotechnology), rabbit anti-mouse myeloperoxidase (MPO) pAbs (Neomarkers, Fremont, CA), rabbit anti-mouse CD3 mAb (Abcam, Tokyo, Japan), cyanine dye 3-conjugated donkey anti-rat IgG pAbs, cyanine dye 3-conjugated donkey anti-goat IgG, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG pAbs, and FITC-conjugated donkey anti-rabbit IgG pAbs (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Immunofluorescence, Staining, Gene Expression, Ligation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Crucial Involvement of IL-6 in Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes

doi: 10.3389/fimmu.2019.03150

Figure Lengend Snippet: The effects of anti-IL-6 antibody (Ab) and recombinant murine IL-6 (rIL-6) in wild-type (WT) mice on thrombus resolution. (A–F) WT mice were intraperitoneally administered with anti-IL-6 as described in section Materials and Methods. (A) Macroscopic appearance of venous thrombi obtained from WT mice treated with anti-IL-6 Ab or control IgG at 10 days after inferior vena cava (IVC) ligation. Representative results from six independent animals are shown here. Thrombus weights (B) and thrombosed blood flow (C) were measured at 10 days after IVC ligation. All values represent the mean ± SEM ( n = 6 animals). * p < 0.05 vs. control IgG. (D–F) Intrathrombotic gene expression of Mmp2 (D) , Mmp9 (E) , and Plau (F) after IVC ligation. Each gene expression was determined by real-time reverse transcription (RT)–PCR as described in section Materials and Methods. All values represent the mean ± SEM ( n = 6 animals). * p < 0.05, ** p < 0.01, control Ig vs. anti-IL-6. (G–L) WT mice were intraperitoneally administered with rIL-6 as described in section Materials and Methods. (G) Macroscopic appearance of venous thrombi obtained from WT mice treated with rIL-6 or phosphate-buffered saline (PBS) at 10 days after IVC ligation. Representative results from six independent animals are shown here. Thrombus weights (H) and thrombosed blood flow (I) were measured at 10 days after IVC ligation. All values represent the mean ± SEM ( n = 6 animals). * p < 0.05, ** p < 0.01, PBS vs. rIL-6. (J–L) Intrathrombotic gene expression of Mmp2 (J) , Mmp9 (K) , and Plau (L) after IVC ligation. Each gene expression was determined by real-time RT-PCR as described in section Materials and Methods. All values represent the mean ± SEM ( n = 6 animals). * p < 0.05, ** p < 0.01, control PBS vs. rIL-6.

Article Snippet: The following mAbs and polyclonal Abs (pAbs) were used for immunohistochemical and double-color immunofluorescence analyses: rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical Company, Japan), mouse anti-IL-6 mAb (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse IL-6 pAbs, rabbit anti-mouse CCL2 pAbs (Novus Biologicals, Centennial, CO), goat anti-mouse MMP-2 pAbs, goat anti-mouse MMP-9 pAbs (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse PLAU (uPA) pAbs, rabbit anti-mouse tPA pAbs, rabbit anti-mouse PAI-1 pAbs (Santa Cruz Biotechnology, Dallas, TX), mouse anti-mouse Col1A2 mAb (Santa Cruz Biotechnology), rabbit anti-mouse myeloperoxidase (MPO) pAbs (Neomarkers, Fremont, CA), rabbit anti-mouse CD3 mAb (Abcam, Tokyo, Japan), cyanine dye 3-conjugated donkey anti-rat IgG pAbs, cyanine dye 3-conjugated donkey anti-goat IgG, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG pAbs, and FITC-conjugated donkey anti-rabbit IgG pAbs (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Control, Ligation, Gene Expression, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Saline, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Crucial Involvement of IL-6 in Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes

doi: 10.3389/fimmu.2019.03150

Figure Lengend Snippet: The effects of recombinant murine IL-6 (rIL-6) on the gene expression of Mmp2, Mmp9 , and Plau and on Stat3 signaling in peritoneal macrophages. Peritoneal macrophages were obtained from wild-type (WT) mice and were stimulated as described in section Materials and Methods. The gene expression of Mmp2 (A) , Mmp9 (B) , and Plau (C) was analyzed by real-time reverse transcription (RT)–PCR. All values represent the mean ± SEM ( n = 6 independent experiments). * p < 0.05, vs. no stimulation. (D) Western blotting analysis using anti-GAPDH pAbs confirmed that an equal amount of protein was loaded onto each lane. Representative results from six independent experiments are shown here. (E) The ratios of p-Stat3/Stat3 were densitometrically determined and are shown. All values represent means ± SEM ( n = 4 independent experiments). (F–H) The effects of anti-IL-6 or Stattic on IL-6-induced gene expression of Mmp2 (F) , Mmp9 (G) , and Plau (H) . Each gene expression was analyzed by real-time RT-PCR. All values represent the mean ± SEM ( n = 4 independent experiments). * p < 0.05; ** p < 0.01, vs. no stimulation.

Article Snippet: The following mAbs and polyclonal Abs (pAbs) were used for immunohistochemical and double-color immunofluorescence analyses: rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical Company, Japan), mouse anti-IL-6 mAb (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse IL-6 pAbs, rabbit anti-mouse CCL2 pAbs (Novus Biologicals, Centennial, CO), goat anti-mouse MMP-2 pAbs, goat anti-mouse MMP-9 pAbs (Santa Cruz Biotechnology, Dallas, Texas), rabbit anti-mouse PLAU (uPA) pAbs, rabbit anti-mouse tPA pAbs, rabbit anti-mouse PAI-1 pAbs (Santa Cruz Biotechnology, Dallas, TX), mouse anti-mouse Col1A2 mAb (Santa Cruz Biotechnology), rabbit anti-mouse myeloperoxidase (MPO) pAbs (Neomarkers, Fremont, CA), rabbit anti-mouse CD3 mAb (Abcam, Tokyo, Japan), cyanine dye 3-conjugated donkey anti-rat IgG pAbs, cyanine dye 3-conjugated donkey anti-goat IgG, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG pAbs, and FITC-conjugated donkey anti-rabbit IgG pAbs (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Gene Expression, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR