Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Ticagrelor inhibits NET release induced by polyP in vitro. ( a ). Fluorescence microscopy for cit-H3/NE staining in control neutrophils treated with synthetic or platelet-derived (natural) polyP, with or without pre-treatment with Ticagrelor or Clopidogrel. One representative out of six independent experiments is shown. Original magnification: 400×, Scale bar: 5μm. Blue: DAPI, Green: NE, Red: cit-H3. ( b ). Percentage of NET-releasing neutrophils as assessed by immunofluorescence. ( c ). MPO-DNA complex levels in NET structures from these stimulations, as assessed by ELISA. Relative integrated optical density was calculated compared to control NETs value. Data from six independent experiments presented as mean ± SD. Statistical significance *, # p < 0.05. All conditions were compared to untreated/control condition and statistical significance is indicated by the symbol “*”. Any further statistical significance of other comparisons is indicated by the symbol “#”. ( d ). Annexin V/Propidium Iodide flow cytometry of control neutrophils in the presence or absence of Ticagrelor/Clopidogrel. One representative out of six independent experiments is shown. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: In Vitro, Fluorescence, Microscopy, Staining, Control, Derivative Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Neutrophils from individuals receiving Ticagrelor were more resistant to NETotic stimulation from polyP. ( a – c ). Fluorescence microscopy for cit-H3/NE staining in neutrophils isolated from a patient with a previous acute coronary syndrome and stent placement that receives Ticagrelor or Clopidogrel as a main antiplatelet treatment and neutrophils from a healthy individual, with or without synthetic polyP. One representative out of five independent experiments is shown. Original magnification: 600×, Scale bar: 5 μm. Blue: DAPI, Green: NE, Red: cit-H3. ( d ). Percentage of NET-releasing neutrophils as assessed by immunofluorescence. ( e ). MPO-DNA complex levels in NET structures from these stimulations, as assessed by ELISA. Data from five independent experiments presented as mean ± SD. Statistical significance * p < 0.05. All conditions were compared to untreated/control condition and statistical significance is indicated by the symbol “*”. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: Fluorescence, Microscopy, Staining, Isolation, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Ticagrelor does not signal through the P2Y12 or the autophagy pathway in neutrophil. ( a ). mRNA fold expression for P2Y12 receptor in control neutrophils treated with synthetic polyP and IRA plasma. Data from three independent experiments presented as mean ± SD. There is no statistical significance. ( b ). Western blot of mTOR p2448 in control neutrophils treated with synthetic polyP, with or without pre-treatment with Ticagrelor. One representative out of four independent experiments is shown. Relative integrated optical density was calculated compared to untreated PMNs value. Data from four independent experiments presented as mean ± SD. Statistical significance * p < 0.05. All conditions were compared to untreated/control conditions and statistical significance is indicated by the symbol “*”. ( c , d ). Confocal microscopy for DAPI/LC3B or DAPI/BECLIN 1 staining in control neutrophils treated with synthetic polyP, with or without pre-treatment with Ticagrelor. One representative out of six independent experiments is shown. Original magnification: 1000×, Scale bar: 5 μm. Blue: DAPI. Red: LC3B. Green: BECLIN 1. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: Expressing, Control, Clinical Proteomics, Western Blot, Confocal Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Τicagrelor is not able to inhibit intracellular expression of tissue factor (TF). ( a ). Fluorescence microscopy for TF/NE staining in control neutrophils treated with IRA plasma, with or without pre-treatment with Ticagrelor or Clopidogrel. One representative out of six independent experiments is shown. Original magnification: 400×, Scale bar: 5 μm. Blue: DAPI, Green: TF, Red: NE. ( b ). Representative fluorescence-activated cell-sorting analysis and ( c ) relative MFIs of TF in control neutrophils treated with IRA plasma, with or without pre-treatment with Ticagrelor or Clopidogrel. Data from six independent experiments presented as mean ± SD. Statistical significance * p < 0.05. n.s.: non-significant. All conditions were compared to the untreated/control condition and statistical significance is indicated by the symbol “*”. Any further non-statistical significance of other comparisons is indicated by the symbol “n.s.”. MFI—mean fluorescence intensity. ( a , b ) One representative out of six independent experiments is shown. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: Expressing, Fluorescence, Microscopy, Staining, Control, Clinical Proteomics, FACS

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Coronary stents induce NET formation. ( a ). Fluorescence microscopy for cit-H3/NE staining in control neutrophils treated with stent and with or without pre-treatment with Ticagrelor or Clopidogrel. One representative out of six independent experiments is shown. Original magnification: 400×, Scale bar: 5 μm. Blue: DAPI, Green: NE, Red: cit-H3. ( b ). MPO-DNA complex levels in NET structures from control neutrophils treated with stent, with or without pre-treatment with Ticagrelor or Clopidogrel, as assessed by ELISA. Relative integrated optical density was calculated when compared to control NETs value. Data from six independent experiments presented as mean ± SD. Statistical significance *, # p < 0.05. All conditions were compared to untreated/control conditions and statistical significance is indicated by the symbol “*”. Any further statistical significance of other comparisons is indicated by the symbol “#”. ( c ). Fluorescence microscopy for TF/NE staining in control neutrophils treated with stent and IRA plasma. One representative out of six independent experiments is shown. Original magnification: 400×, Scale bar: 5 μm. Blue: DAPI, Green: TF, Red: NE. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: Fluorescence, Microscopy, Staining, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: C5a promoted arterial thrombosis by triggering neutrophils to release NETs. A , B H&E staining of thrombus, cross-sections ( A ) and longitudinal sections ( B ). Scale bar = 100 µm. C Quantification of the thrombus size in cross-sections by H&E staining ( n = 5 each). D Thrombus weight ( n = 5 each). E The blood flow velocity in the LICA was dramatically decreased in the FeCl 3 -induced thrombus mice compared with the sham mice, and this change was reversed by PMX53. F Quantification of the blood flow velocity ( n = 4 each). G Immunofluorescence staining of cross-sections of the LCCA for CitH3 (red), Ly6g (green) and DAPI (blue) 6 h after FeCl 3 exposure. Thirty minutes before FeCl 3 exposure, PMX53 was administered by intraperitoneal injection. Scale bar = 75 µm. H Quantification of NET formation capacity shown as the ratio of NETs/neutrophil in LCCA cross-sections subjected to immunofluorescence staining (NaCl group = 3, PMX53 group = 4). I Representative images of immunofluorescence stainings of NET formation for DNA (SYTOX green), CitH3 (red), Ly6g(blue) in vitro after stimulation of C5a, and NET formation was attenuated by PMX53. Scale bar = 75 µm. J Quantification of NET formation capacity in vitro shown as the percentage of NET release, which was assessed by immunofluorescence staining ( n = 3 each). (K) NETosis was measured using a plate reader assay. NET release is expressed as percentage of total DNA ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Staining, Immunofluorescence, Injection, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Interactions among C5a, mitochondrial STAT3 and NETs. A , B Western blot analysis was performed to test the expression levels of mitochondrial STAT3 and p-STAT3 (Ser 727 ) in neutrophil-like cells cocultured with C5a, compared with the control. VDAC, a marker of mitochondria, was used as a loading control for mitochondria ( n = 5 each). C NET release in response to buffer or AG490 was measured using a plate reader assay ( n = 3 each). D Representative images of immunofluorescence staining for DNA (SYTOX green), CitH3 (red) and Ly6g (blue) in vitro after stimulation with AG490 showing the presence of NETs. Scale bar = 100 µm. E Quantification of NET formation capacity shown as the percentage of NET release in vitro after stimulation with buffer or AG490, as assessed by immunofluorescence staining. ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Marker, Immunofluorescence, Staining, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: The reduction in arterial thrombotic burden induced by PMX53 was abolished by AG490 in vivo. A , B H&E staining of thrombus, cross-sections A and longitudinal sections B . The thrombus area was reduced by PMX53, and AG490 abolished this effect. A Scale bar = 100 µm; B Scale bar = 200 µm. C Quantification of the thrombus size in cross-sections by H&E staining ( n = 5 each). D Thrombus weight ( n = 5 each). E Blood flow velocity in the LICA. AG490 reversed the increased in blood flow induced by PMX53. F Quantification of the blood flow velocity ( n = 4 each). G Immunofluorescence staining of cross-sections of the LCCA for CitH3 (red), Ly6g (green) and DAPI (blue) 6 h after FeCl 3 exposure. Scale bar = 75 µm. H Quantification of NET formation capacity shown as the ratio of NETs/neutrophil in LCCA cross-sections subjected to immunofluorescence staining (NaCl group = 3; PMX53 group = 4; PMX53 + AG490 group = 3; AG490 group = 4). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: In Vivo, Staining, Immunofluorescence

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Visual summary: the effect of complement C5a in arterial thrombosis. In arterial thrombosis, C5a chemotactically attracts neutrophils to migrate towards the culprit site and triggers the release of NETs, which contribute to thrombosis by promoting coagulation and stabilizing clots. C5a-induced promotion of NET release is dependent on Mito-ROS production. C5a induces the Mito-ROS production by inhibiting mitochondrial STAT3 activity

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Coagulation, Activity Assay

Journal: Journal of Clinical Medicine

Article Title: Immunohistochemical Results of Soft Tissues Around a New Implant Healing-Abutment Surface: A Human Study

doi: 10.3390/jcm9041009

Figure Lengend Snippet: Expression pattern of inflammation and junctional markers. Statistically significant differences between different surface treatment were found ( p < 0.001).

Article Snippet: Briefly, samples were then incubated with anti-occludin antibody 5 µg/mL (Abcam Cambridge, UK) or human neutrophil defensins (alpha-defensins, HNP1–3) (Hycult Biotech, Uden, The Netherlands) for 1 h. Successively slides were incubated with goat anti polyvalent antibody for 10 min and subsequently with peroxidase for 10 min. After incubation samples were washed and treated by 3,3’-Diaminobenzidine DAB substrate.

Techniques: Expressing

Journal: Journal of Clinical Medicine

Article Title: Immunohistochemical Results of Soft Tissues Around a New Implant Healing-Abutment Surface: A Human Study

doi: 10.3390/jcm9041009

Figure Lengend Snippet: Immunostaining against HNP1–3 of the region of the gingiva adherent to the laser-treated ( A ), to the point of passage between the two surfaces ( B ) (dotted blue line represents an arbitrary hypothetical line) and to machined ( C ) surfaces, as indicated. Magnification 10X. ( D ) Percentage of HNP1–3 immunostaining positive area in the lamina propria and in the epithelial layers, as indicated. Values are expressed as mean ±SD of five determinations in randomly chosen sections for each specimen. * indicates values statistically different ( p < 0.001) between region of the gingiva adherent to the laser-treated- or to machined surfaces, as indicated.

Article Snippet: Briefly, samples were then incubated with anti-occludin antibody 5 µg/mL (Abcam Cambridge, UK) or human neutrophil defensins (alpha-defensins, HNP1–3) (Hycult Biotech, Uden, The Netherlands) for 1 h. Successively slides were incubated with goat anti polyvalent antibody for 10 min and subsequently with peroxidase for 10 min. After incubation samples were washed and treated by 3,3’-Diaminobenzidine DAB substrate.

Techniques: Immunostaining

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: Lipocalin 2 is inducible in HBE. Air-liquid interface cultures of HBE1 (A) or NHBE (B) were stimulated on the basolateral surface with IL-17A, IL-17F, or both, with and without synergistic TNF-α. In each panel, Western blot analysis of cell lysates using monoclonal anti-lipocalin 2 detects a 25-kDa protein shown in the top blot. Bottom blot in each section shows the results of probing the same blot with anti-GAPDH. Densitometry results from Western blots run on three replicates (A) or three patients (B) are above each gel in both panels. In each set, relative intensities were determined by taking absolute intensities quantified from the Western blot and dividing by the absolute intensity of the unstimulated lane on that blot.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Western Blot

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: Non-aerobactin producing KP strain is inhibited by lipocalin 2. A, KP ATCC strain 43816 was seeded into wells of a 96-well plate and cultured in the presence of increasing concentrations of recombinant lipocalin 2. OD600 readings from triplicate wells of each concentration were taken as shown. Growth inhibition is seen starting at 10 µM. B, Lipocalin 2 effect is reversed with iron repletion. Assay above was repeated with 40 µM recombinant lipocalin 2 and increasing concentrations of FeCl3 in the medium. Final OD600 was taken after 15 h of culture. Results reflect averages of triplicate wells.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Cell Culture, Recombinant, Concentration Assay, Inhibition

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: Lipocalin 2 is up-regulated in the lung during KP infection in CCSP+ and CCSP− cells. A, Twelve C57BL/6 (WT) mice were infected with KP and four mice per cohort were sacrificed at 0, 4, and 16 h after infection. Top panel, Lung homogenates were analyzed by Western blot using anti-lipocalin 2. Bottom panel, Western blot was reprobed with Ab against GAPDH. Increased lipocalin 2 levels are seen as early as 4 h after infection and persist to 16 h. B, Immunohistochemical analysis of lung sections using anti-lipocalin 2 shows lipocalin 2-positive cells as early as 4 h after infection. This finding was reproduced for three animals. C, Dual immunofluorescence analysis using anti-lipocalin 2 and anti-CCSP Abs shows that lipocalin 2 is present in both CCSP+ and CCSP− cells.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Infection, Western Blot, Immunohistochemical staining, Immunofluorescence

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: Lipocalin 2 up-regulation is TLR4 dependent. Littermate controls (□) and KO mice (■) were infected and sacrificed after 4 h, n = 4–6 animals/cohort. Lung homogenates were analyzed by ELISA for lipocalin 2 levels. TLR4 KO animals are unable to up-regulate lipocalin 2 protein levels after 4 h of infection, while p19 KO, p35 KO, and p40 KO animals retained this ability.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: TLR4 regulation of lipocalin 2 is largely MyD88 dependent. TLR4 KO, MyD88 KO, Trif KO mice, and their strain controls (WT) were infected and sacrificed after 4 h, n = 4–6 animals/cohort. Immunohistochemistry results were reproduced for three animals. Lungs were fixed in formalin and paraffin embedded for immunohistochemical analysis or obtained for homogenization for ELISA analysis. A, Results of staining for lipocalin 2 in the TLR4 KO at 4-h infection showing no discernible lipocalin 2 signal. B, Lipocalin 2 ELISA results from lung homogenates obtained after 4 h of infection. Lipocalin 2 is diminished in both the MyD88 KO and Trif KO mice but the levels are more profoundly affected by MyD88 deficiency.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Infection, Immunohistochemistry, Immunohistochemical staining, Homogenization, Enzyme-linked Immunosorbent Assay, Staining

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: Th17 cytokines are not necessary for lipocalin 2 induction. A, Six strain controls (WT) and six IL-17R KO mice were infected and two animals of each genotype were sacrificed at 0, 4, and 16 h of infection. Western blot analysis was performed on lung homogenates. Top panel shows anti-lipocalin 2 probe with anti-GAPDH-loading control. Bottom panel shows ELISA results of the same infection repeated with three animals per cohort. B, Strain controls (WT) and IL-17A KO animals were subjected to infection and sacrificed at 16 and 24 h (four animals per cohort). Lung homogenates were analyzed for lipocalin 2 levels by ELISA. C, Six C57BL/6 (WT) animals were given neutralizing doses of anti-IL-22 (three animals) or IgG control (three animals) and infected and sacrificed after 4 h of infection. Lung homogenates were analyzed for lipocalin 2 levels by ELISA.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: IL-1β induces lipocalin 2 and rescues TLR4 KO from KP infection in an IFN-γ-independent manner. A, MLE were stimulated in triplicate with IL-1β with or without TNF-α. Cell lysates were assayed for lipocalin 2 levels by ELISA. B, Three strain controls (WT) and three IL-1R KO mice were infected and lung homogenates were analyzed for lipocalin 2 levels. IL-1R KO mice show decreased levels of lipocalin 2 after infection. C, Six WT and six TLR4 KO mice were infected and another cohort of TLR4 KO mice (six animals) was given IL-1β. Animals were sacrificed at 12 h of infection and lung homogenates were assayed for lipocalin 2. Lipocalin 2 levels in the TLR4 KO again were significantly diminished compared with WT and significantly increased in TLR4 KO mice that received IL-1β. D, TLR4 KO mice receiving IL-1β were rescued from infection as demonstrated by bacterial load after 12 h of infection. E, WT mice up-regulate IFN-γ during infection, but TLR4 KO, including TLR4 KO receiving IL-1β do not; therefore, the mechanism of IL-1β - mediated rescue from infection is not due to IFN-γ up-regulation. *, Statistical significance compared with unstimulated or WT and **, statistical significance compared with untreated KO.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: Recombinant lipocalin 2 rescues TLR4 KO from KP infection. Two Lcn2 KO cohorts (six mice per group) and their strain controls (six mice) were infected with KP as before. One Lcn2 KO cohort was given recombinant lipocalin 2 while the other two cohorts received PBS plus BSA. Mice were sacrificed at 12 h of infection and lung homogenates were plated to determine CFU. Reconstitution of the TLR4 KO results in statistically significant reduction in lung bacterial load and spleen dissemination of KP. *, Statistical significance compared with WT and **, statistical significance compared with untreated KO.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Recombinant, Infection

Journal:

Article Title: Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection 1

doi: 10.4049/jimmunol.0803282

Figure Lengend Snippet: Recombinant lipocalin 2 rescues Lcn2 KO from KP infection. A, Lcn2 KO mice were infected and are susceptible as demonstrated by bacterial loads in lung homogenates after 4 and 12 h of infection (four animals per cohort). B, Extrapulmonary dissemination of KP to the spleen is also increased in the same Lcn2 KO animals compared with controls at 12 h of infection. C, Reconstituting Lcn2 KO mice with recombinantly expressed lipocalin 2 protein (Lcn 2) results in statistically significant reduction in lung bacterial load at 12 h of infection in these mice compared with Lcn2 KO mice that did not receive Lcn 2 (six animals per cohort). D, Wet:dry ratios in Lcn2 KO mice receiving Lcn 2 are significantly lower compared with Lcn2 KO mice that did not receive Lcn 2. Data reflect six mice per group. *, Statistical significance compared with WT and **, statistical significance compared with untreated KO.

Article Snippet: Lung sections were stained per standard protocols ( 21 ) using affinity-purified goat anti-mouse lipocalin 2 (R&D Systems) followed by HRP-conjugated rabbit anti-goat IgG (Pierce) and development using a HRP chromogen kit (Abcam) for immunohistochemical sections.

Techniques: Recombinant, Infection

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Histones and Neutrophil Extracellular Traps Enhance Tubular Necrosis and Remote Organ Injury in Ischemic AKI

doi: 10.1681/ASN.2016080925

Figure Lengend Snippet: NETs in the mouse kidney with acute injury. (A) Representative NETs staining in outer medullary lesions of unilateral IRI kidney (ischemia for 35 minutes and reperfusion for 24 hours). Colocalization of CitH3 (red), Ly6b (green), and swelled nuclei (blue) surrounding tubular duct indicates the NETs formation. Scale bar, 50 μm. (B) Histology of unilateral IRI kidney at different time points after reperfusion and different ischemia times. (Row 1) Ly6b (green). (Row 2) CitH3 (red). (Row 3) TUNEL (green). (Row 4) Periodic acid–Schiff (PAS) staining. Scale bar, 200 μm. (C) Ly6b-positive area, (D) CitH3-positive area, (E) TUNEL-positive area, and (F) histologic evaluation of PAS staining at different times. (G) Representative image and (H) tubular injury score at different ischemic times. (I) CitH3 expression of unilateral IRI kidney by immunoblotting. Data are mean±SEM from five mice in each group.

Article Snippet: For immunostaining, rat anti-mouse Ly6b (neutrophils) Ab (AbD Serotec, Oxford, United Kingdom), rabbit anti-CitH3 Ab (Abcam, Cambridge, United Kingdom), goat anti-NE Ab (Santa Cruz Biotechnology), and rabbit anti-KIM1 Ab (Abcam) were used.

Techniques: Staining, TUNEL Assay, Expressing, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Histones and Neutrophil Extracellular Traps Enhance Tubular Necrosis and Remote Organ Injury in Ischemic AKI

doi: 10.1681/ASN.2016080925

Figure Lengend Snippet: AKI-related remote organ injury is caused by circulating NETs and DAMPs, such as histones. (A) Plasma TNF-α, (B) IL-6, and (C) histone 3 content in sham-operated mice and bilateral IRI kidney mice (ischemia for 35 minutes and reperfusion for 24 hours) was measured by (A and B) ELISA and (C) immunoblotting methods. As a positive control for plasma histone, the plasma of LPS-induced sepsis mice was used. (D) Tissue injury, (E) neutrophil infiltration, and (F) NETs expression in multiorgan (kidney, lung, liver, brain, heart, and pancreas) sham and bilateral IRI (ischemia for 35 minutes and reperfusion for 24 hours) kidney mice were evaluated by TUNEL staining, Ly6b immunostaining, and immunoblotting, respectively. (G) Lung injury followed by bilateral IRI kidney (ischemia for 35 minutes and reperfusion for 24 hours) treated with vehicle, PAD inhibitor, neutrophil depletion, necrosis inhibitor, necrosis inhibitor and PAD inhibitor, or aHisAbs was evaluated by (upper panel) NETs immunostaining (blue, 49,6-diamidin-2-phenylindol [DAPI]; green, NE; red, CitH3) and (lower panel) TUNEL staining (lower figures). The graphs show (H) NETs area and (I) TUNEL-positive area in lung. (J) The cell number in BAL of these groups was counted. (K–M) The quantification of TUNEL-positive area in (K) liver, (L) heart, and (M) brain in each group. (N) Plasma TNF-α and (O) IL-6 in differently treated mice were measured by ELISA method. Data show the mean±SEM from at least five mice in each group. Scale bar, 100 μm. *P<0.05 versus respective control; **P<0.01 versus respective control; ***P<0.01 versus respective control; #P<0.05 compared with aHisAbs group.

Article Snippet: For immunostaining, rat anti-mouse Ly6b (neutrophils) Ab (AbD Serotec, Oxford, United Kingdom), rabbit anti-CitH3 Ab (Abcam, Cambridge, United Kingdom), goat anti-NE Ab (Santa Cruz Biotechnology), and rabbit anti-KIM1 Ab (Abcam) were used.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Positive Control, Expressing, TUNEL Assay, Staining, Immunostaining, Control

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat anti-NIMP-R14 , Santa Cruz Biotechnology , Cat# sc-59338 RRID: AB_2167795.

Techniques: Recombinant, Software, Microscopy