rabbit anti bovine serum albumin antibody  (Valiant)

 
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    Name:
    Anti bovine serum rabbit antiserum
    Description:

    Catalog Number:
    08650011
    Price:
    103.3
    Category:
    Life Sciences Antibodies Antisera
    Applications:
    Immunohistochemistry, Immunoelectrophoresis (IE) , Immunoelectrophoresis
    Size:
    2 mL
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    Structured Review

    Valiant rabbit anti bovine serum albumin antibody
    Anti bovine serum rabbit antiserum

    https://www.bioz.com/result/rabbit anti bovine serum albumin antibody/product/Valiant
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti bovine serum albumin antibody - by Bioz Stars, 2021-05
    85/100 stars

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    Mouse Assay:

    Article Title: Persistent neutrophil dysfunction and suppression of acute lung injury in mice following cecal ligation and puncture sepsis
    Article Snippet: .. Briefly, following anesthesia with ketamine and xylazine, mice received 125 μg of rabbit anti-bovine serum albumin antibody (MP Biomedicals, Solon, OH) during inspiration in a volume of 30 μl saline. .. Then, 1 mg of BSA (Sigma) was injected i.v. in 200 μl saline (sham mice received saline only i.v.).

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  • 94
    Valiant anti bsa igg
    Effects of C/EBPβ deficiency on <t>IgG</t> immune complex-induced acute lung injury 4 h after IgG immune complex deposition, BAL fluids and whole lungs were harvested. A , Mouse albumin content in BAL fluids was determined using ELISA as an index of lung microvascular permeability. B , Changes in lung MPO activity was measured. C and D , Total cell and neutrophil accumulation in BAL fluids were counted. E , Lung sections were stained with H E (40 × magnification). Lung sections shown included: C/EBPβ +/+ + <t>anti-BSA,</t> C/EBPβ +/+ + IgG IC, C/EBPβ +/− + anti-BSA, C/EBPβ +/− + IgG IC, C/EBPβ −/− + anti-BSA, and C/EBPβ −/− + IgG IC. Results are means ± S. E. M. for 3 (control group) or 5 (IgG immune complex-challenged group) mice for each group.
    Anti Bsa Igg, supplied by Valiant, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti bsa igg/product/Valiant
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti bsa igg - by Bioz Stars, 2021-05
    94/100 stars
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    91
    Valiant rabbit anti hrp
    Proximal localization of Shot and its mediation of T antigen trafficking specifically to the proximal segment. ( a – a” ) Localization of T antigens, Shot, and <t>HRP</t> antigens in a permeabilized wild-type primary cultured neuron. Similar to T antigens, Shot was localized in the proximal axon segment in 40% of the permeabilized neurons ( a’ ). The intra-axonal boundary (filled arrowheads) defined by localization of Shot was consistent with that defined by T antigens. ( b ) Fluorescence intensity of T antigens and Shot along axons. The filled arrowhead indicates the intra-axonal boundary. ( c – c” ) Localization of T antigens, <t>ROBO3,</t> and HRP antigens on the surface of a wild-type neuron. T antigens are localized on the surface of the proximal axon segment. Conversely, ROBO3 is localized on the surface of the distal axon segment. Filled and open arrowheads indicate the intra-axonal boundaries defined by the localization of T antigens and ROBO3, respectively ( c , d ). ( d ) Fluorescence intensity of T antigens, ROBO3, and HRP antigens along axons. ( e – e” ) Localization of T antigens, Shot, and HRP antigens in a wild-type embryo at stage 16. T antigens and Shot were both expressed in the CNS and in the PNS. ( f , f’ ) Localization of T and HRP antigens on the surface of a shot 3 / shot SF20 transheterozygous mutant neuron. ( g , g’ ) Localization of T and HRP antigens in a shot 3 / shot SF20 mutant permeabilized neuron. ( h ) Histogram showing the localization of T antigens in/on the wild-type and shot 3 / shot SF20 mutant neurons. The histogram of the surface expression for the wild-type is the same as that at 24 h (shown in Fig. 1h ). In the shot 3 / shot SF20 mutant, the percentage of neurons with proximal localization of T antigens (green) was significantly decreased. P -values were calculated using the G-test. ** p = 3.3 × 10 −14 , * p = 1.8 × 10 −12 . Scale bar: 10 μm ( a , c , f , g ), 50 μm ( e ).
    Rabbit Anti Hrp, supplied by Valiant, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti hrp/product/Valiant
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti hrp - by Bioz Stars, 2021-05
    91/100 stars
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    N/A
    Product is the lyophilized powder of rabbit IgG fraction to bovine albumin and buffer salts Rabbit IgG fraction to bovine albumin is used as a reagent in immunohistochemistry and immunoblotting
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    Effects of C/EBPβ deficiency on IgG immune complex-induced acute lung injury 4 h after IgG immune complex deposition, BAL fluids and whole lungs were harvested. A , Mouse albumin content in BAL fluids was determined using ELISA as an index of lung microvascular permeability. B , Changes in lung MPO activity was measured. C and D , Total cell and neutrophil accumulation in BAL fluids were counted. E , Lung sections were stained with H E (40 × magnification). Lung sections shown included: C/EBPβ +/+ + anti-BSA, C/EBPβ +/+ + IgG IC, C/EBPβ +/− + anti-BSA, C/EBPβ +/− + IgG IC, C/EBPβ −/− + anti-BSA, and C/EBPβ −/− + IgG IC. Results are means ± S. E. M. for 3 (control group) or 5 (IgG immune complex-challenged group) mice for each group.

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

    Article Title: Critical Role for CCAAT/Enhancer Binding Protein ? in Immune Complex-Induced Acute Lung Injury

    doi: 10.4049/jimmunol.1200877

    Figure Lengend Snippet: Effects of C/EBPβ deficiency on IgG immune complex-induced acute lung injury 4 h after IgG immune complex deposition, BAL fluids and whole lungs were harvested. A , Mouse albumin content in BAL fluids was determined using ELISA as an index of lung microvascular permeability. B , Changes in lung MPO activity was measured. C and D , Total cell and neutrophil accumulation in BAL fluids were counted. E , Lung sections were stained with H E (40 × magnification). Lung sections shown included: C/EBPβ +/+ + anti-BSA, C/EBPβ +/+ + IgG IC, C/EBPβ +/− + anti-BSA, C/EBPβ +/− + IgG IC, C/EBPβ −/− + anti-BSA, and C/EBPβ −/− + IgG IC. Results are means ± S. E. M. for 3 (control group) or 5 (IgG immune complex-challenged group) mice for each group.

    Article Snippet: Anti-BSA IgG was purchased from MP Biomedicals (Solon, OH).

    Techniques: Enzyme-linked Immunosorbent Assay, Permeability, Activity Assay, Staining, Mouse Assay

    Western blot analysis for tyrosine-phosphorylated (PY) FcRγ-subunit of PMN or macrophage lysates after in vitro incubation. (A,B) 3×10 6 cells/ml from either Wt or TLR4 mut mice were incubated for 5, 15, and 30 min with IgG immune complexes (IgGIC; 100 µg/ml). (C,D) The same protocol was used for stimulation with LPS (20 ng/ml). (E) Lysates from either Wt or TLR4 mut mice that were incubated with polymyxin-treated BSA immune complexes (100 µg/ml) under the same conditions as described above. Corresponding loading controls are displayed in the lower panels.

    Journal: PLoS Pathogens

    Article Title: Cross-Talk between TLR4 and Fc?ReceptorIII (CD16) Pathways

    doi: 10.1371/journal.ppat.1000464

    Figure Lengend Snippet: Western blot analysis for tyrosine-phosphorylated (PY) FcRγ-subunit of PMN or macrophage lysates after in vitro incubation. (A,B) 3×10 6 cells/ml from either Wt or TLR4 mut mice were incubated for 5, 15, and 30 min with IgG immune complexes (IgGIC; 100 µg/ml). (C,D) The same protocol was used for stimulation with LPS (20 ng/ml). (E) Lysates from either Wt or TLR4 mut mice that were incubated with polymyxin-treated BSA immune complexes (100 µg/ml) under the same conditions as described above. Corresponding loading controls are displayed in the lower panels.

    Article Snippet: Immune Complex Lung Injury To induce IgGIC lung injury, tracheae of mice were surgically exposed and 125 µg rabbit anti-BSA IgG (MP Biomedicals) was administered using a 30 gauge needle (volume of 42 µl/mouse) followed by intravenous injection of BSA (500 µg; Sigma).

    Techniques: Western Blot, In Vitro, Incubation, Mouse Assay

    Parameters of acute lung injury in Wt and TLR4 mut mice. (A) Lung injury (as measured by leak of 125 I-BSA into lung) in Wt, TLR4 mut, TLR4 +/+ , and TLR4 −/− mice receiving LPS intratracheally. (B) Permeability indices in Wt, TLR4 mut, TLR4 +/+ , and TLR4 −/− mice after intrapulmonary immune complex formation following administration of BSA (i.v.) and anti-BSA IgG (i.t.). (C) IL-6 levels in BAL fluids after IgG immune complex (IgGIC)- or LPS-induced lung injury using Wt and TLR4 mut mice. (D) TNFα in BAL fluids from the same mice described in frame (C). For each bar, n≥5. (E) Lung injury induced by IgG immune complexes (IgGIC) in Wt and TLR4 mut mice after endotoxin removal by polymyxin. (F) Lung permeability after intratracheal (i.t.) administration of anti-BSA IgG and intravenous (i.v.) injection of BSA, PBS i.t., and BSA i.v. or anti-BSA i.t. and PBS i.v. (G) IgGIC-induced lung injury in FcRγ-subunit −/− mice in comparison to Wt mice (FcRγ-subunit +/+ ). For each bar, n≥5.

    Journal: PLoS Pathogens

    Article Title: Cross-Talk between TLR4 and Fc?ReceptorIII (CD16) Pathways

    doi: 10.1371/journal.ppat.1000464

    Figure Lengend Snippet: Parameters of acute lung injury in Wt and TLR4 mut mice. (A) Lung injury (as measured by leak of 125 I-BSA into lung) in Wt, TLR4 mut, TLR4 +/+ , and TLR4 −/− mice receiving LPS intratracheally. (B) Permeability indices in Wt, TLR4 mut, TLR4 +/+ , and TLR4 −/− mice after intrapulmonary immune complex formation following administration of BSA (i.v.) and anti-BSA IgG (i.t.). (C) IL-6 levels in BAL fluids after IgG immune complex (IgGIC)- or LPS-induced lung injury using Wt and TLR4 mut mice. (D) TNFα in BAL fluids from the same mice described in frame (C). For each bar, n≥5. (E) Lung injury induced by IgG immune complexes (IgGIC) in Wt and TLR4 mut mice after endotoxin removal by polymyxin. (F) Lung permeability after intratracheal (i.t.) administration of anti-BSA IgG and intravenous (i.v.) injection of BSA, PBS i.t., and BSA i.v. or anti-BSA i.t. and PBS i.v. (G) IgGIC-induced lung injury in FcRγ-subunit −/− mice in comparison to Wt mice (FcRγ-subunit +/+ ). For each bar, n≥5.

    Article Snippet: Immune Complex Lung Injury To induce IgGIC lung injury, tracheae of mice were surgically exposed and 125 µg rabbit anti-BSA IgG (MP Biomedicals) was administered using a 30 gauge needle (volume of 42 µl/mouse) followed by intravenous injection of BSA (500 µg; Sigma).

    Techniques: Mouse Assay, Permeability, Injection

    Association between TLR4 and FcRγIII. Peritoneal PMNs and macrophages (3×10 6 cells/ml) from Wt mice and FcRγ-subunit −/− mice were incubated in vitro for 30 min with either IgG immune complexes (IgGIC; 100 µg/ml), LPS (20 ng/ml), or the combination. (A,B) Western blot analysis (IB) for FcγRIII of Wt PMN or macrophage lysates co-immunoprecipitated (IP) with anti-TLR4. (C,D) Reverse direction immunoprecipitation using anti-FcγRII/III IgG followed by Western blot analysis for TLR4. (E,F) Western blot analysis for FcγRIII of PMNs or macrophages from FcγRIII −/− co-immunoprecipitated (IP) with anti-TLR4. (G,H) Samples were immunoprecipitated with anti-TLR6 IgG and probed for FcγRIII. (I,J) Immunoprecipitation with anti-CD23 followed by Western blots using anti-TLR4 IgG. (K) Western blots (IB) of cell lysates of Wt macrophages that were incubated for 30 min with BSA IgG immune complexes (IgGIC; 100 µg/ml), polymyxin-treated BSA IgG immune complexes (p.-t. BSA IC; 100 µg/ml) or peroxidase/anti-peroxidase IgGIC immune complexes (PAP IC, 100 µg/ml). IB for FcγRIII of Wt macrophage lysates co-immunoprecipitated (IP) with anti-TLR4. Corresponding loading controls are displayed in lower panels.

    Journal: PLoS Pathogens

    Article Title: Cross-Talk between TLR4 and Fc?ReceptorIII (CD16) Pathways

    doi: 10.1371/journal.ppat.1000464

    Figure Lengend Snippet: Association between TLR4 and FcRγIII. Peritoneal PMNs and macrophages (3×10 6 cells/ml) from Wt mice and FcRγ-subunit −/− mice were incubated in vitro for 30 min with either IgG immune complexes (IgGIC; 100 µg/ml), LPS (20 ng/ml), or the combination. (A,B) Western blot analysis (IB) for FcγRIII of Wt PMN or macrophage lysates co-immunoprecipitated (IP) with anti-TLR4. (C,D) Reverse direction immunoprecipitation using anti-FcγRII/III IgG followed by Western blot analysis for TLR4. (E,F) Western blot analysis for FcγRIII of PMNs or macrophages from FcγRIII −/− co-immunoprecipitated (IP) with anti-TLR4. (G,H) Samples were immunoprecipitated with anti-TLR6 IgG and probed for FcγRIII. (I,J) Immunoprecipitation with anti-CD23 followed by Western blots using anti-TLR4 IgG. (K) Western blots (IB) of cell lysates of Wt macrophages that were incubated for 30 min with BSA IgG immune complexes (IgGIC; 100 µg/ml), polymyxin-treated BSA IgG immune complexes (p.-t. BSA IC; 100 µg/ml) or peroxidase/anti-peroxidase IgGIC immune complexes (PAP IC, 100 µg/ml). IB for FcγRIII of Wt macrophage lysates co-immunoprecipitated (IP) with anti-TLR4. Corresponding loading controls are displayed in lower panels.

    Article Snippet: Immune Complex Lung Injury To induce IgGIC lung injury, tracheae of mice were surgically exposed and 125 µg rabbit anti-BSA IgG (MP Biomedicals) was administered using a 30 gauge needle (volume of 42 µl/mouse) followed by intravenous injection of BSA (500 µg; Sigma).

    Techniques: Mouse Assay, Incubation, In Vitro, Western Blot, Immunoprecipitation

    In vitro cytokine responses of elicited peritoneal PMNs and macrophages to LPS and IgGIC. In vitro cytokine responses of elicited peritoneal PMNs (A–D) and macrophages (E,F). Cells (3×10 6 cells/ml) from either Wt or TLR4 mut mice were incubated for 4 hr with LPS (20 ng/ml) or IgGIC; 100 µg/ml), respectively. In addition, macrophages were incubated with polymyxin-treated BSA IgG immune complexes (p.-t. BSA IC, 100 µg/ml) or peroxidase/anti-peroxidase IgGIC immune complexes (PAP IC, 100 µg/ml). (A) IL-6 release from PMNs after LPS stimulation. (B) TNFα levels after incubation of PMNs with LPS. (C) Concentration of IL-6 in supernatants when PMNs were exposed to IgGIC. (D) Production of TNFα by PMNs and macrophages in the presence of IgGIC. Ctrl = control levels of non-stimulated cells. (E) Release of IL-6 by macrophages into supernatant fluids after stimulation with LPS, IgGIC, p.-t. BSA IC, or PAP IC. (F) TNFα production by macrophages exposed to LPS, IgGIC, p.-t. BSA IC, or PAP IC. The experiments were performed in triplicates for each condition (each bar) with n≥3 donors of cells for each mouse strain, Wt or TLR4 mut. Differences between controls and stimulated cells were—if not otherwise noted—statistically significant (p

    Journal: PLoS Pathogens

    Article Title: Cross-Talk between TLR4 and Fc?ReceptorIII (CD16) Pathways

    doi: 10.1371/journal.ppat.1000464

    Figure Lengend Snippet: In vitro cytokine responses of elicited peritoneal PMNs and macrophages to LPS and IgGIC. In vitro cytokine responses of elicited peritoneal PMNs (A–D) and macrophages (E,F). Cells (3×10 6 cells/ml) from either Wt or TLR4 mut mice were incubated for 4 hr with LPS (20 ng/ml) or IgGIC; 100 µg/ml), respectively. In addition, macrophages were incubated with polymyxin-treated BSA IgG immune complexes (p.-t. BSA IC, 100 µg/ml) or peroxidase/anti-peroxidase IgGIC immune complexes (PAP IC, 100 µg/ml). (A) IL-6 release from PMNs after LPS stimulation. (B) TNFα levels after incubation of PMNs with LPS. (C) Concentration of IL-6 in supernatants when PMNs were exposed to IgGIC. (D) Production of TNFα by PMNs and macrophages in the presence of IgGIC. Ctrl = control levels of non-stimulated cells. (E) Release of IL-6 by macrophages into supernatant fluids after stimulation with LPS, IgGIC, p.-t. BSA IC, or PAP IC. (F) TNFα production by macrophages exposed to LPS, IgGIC, p.-t. BSA IC, or PAP IC. The experiments were performed in triplicates for each condition (each bar) with n≥3 donors of cells for each mouse strain, Wt or TLR4 mut. Differences between controls and stimulated cells were—if not otherwise noted—statistically significant (p

    Article Snippet: Immune Complex Lung Injury To induce IgGIC lung injury, tracheae of mice were surgically exposed and 125 µg rabbit anti-BSA IgG (MP Biomedicals) was administered using a 30 gauge needle (volume of 42 µl/mouse) followed by intravenous injection of BSA (500 µg; Sigma).

    Techniques: In Vitro, Mouse Assay, Incubation, Concentration Assay

    Arp2/3-mediated actin polymerization and myosin-II have distinct roles in phagocytic force generation and progression. a, Confocal images of drug-treated fixed RAW cells phagocytosing DAAM-particles functionalized with AF488-Cadaverine, BSA and anti-BSA IgG. Cells were treated with DMSO, CK666 (150 μM), Blebbistatin (15 μM) and SMIFH2 (10 μM) for 30 minutes prior to phagocytic challenge. Each target is approximately 60% engulfed. Fixed cells were stained for F-actin, and particles were labelled with a fluorescent secondary antibody to reveal the exposed surface. Left column: composite maximum intensity projections (MIP) of confocal z-stacks, 2 nd -3 rd column: single confocal slices through particle centroid. Scale bar, 5μm. b, Particle shape reconstructions from a, revealing detailed target deformations and localization of F-actin over the particle surface. Stars mark the base of the phagocytic cup, and the phagocytic axis is horizontal. Scale bars, 3 μm. c, Normal and shear stresses exerted on the target. Negative normal forces denote (inward) compressive forces. d, Average deformation and F-actin intensity profiles along the phagocytic axis to the cup rim. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets in 30 bins. Targets before 40% engulfment were excluded. e, f, Violin plots of all measured particles, showing individual phagocytic events (colored markers), mean (black cross) and median (dashed line). e, F-actin peak intensity and width. f, F-actin intensity in the cup (behind the rim), measured right (3μm) behind the main peak for each particle. g, Upper panel, cumulative distribution function of the engulfment stage of randomly selected phagocytic events (n = 68, 63, 73 55 respectively) from 3 independent experiments. Two sample Kolmogorov-Smirnov test was used (p = 0.016*). Lower panel, fraction late-stage cups. Error bars indicate st.d. estimated by treating phagocytosis as a Bernoulli process. Fisher’s exact test was used to compare fractions (p = 1.9×10 −4 )***. h, Sphericity and i, constriction magnitude of DAAM particle changes with phagocytic progression upon drug treatment. Colored markers indicate individual events, black lines indicate averages within 5 bins. Right column, violin plots of all events. Marker and line styles as in e. All statistical tests were two-side Wilcoxon rank sum test comparing with the DMSO control (gray) over the same bin with significance levels: p

    Journal: bioRxiv

    Article Title: Phagocytic “teeth” and myosin-II “jaw” power target constriction during phagocytosis

    doi: 10.1101/2021.03.14.435346

    Figure Lengend Snippet: Arp2/3-mediated actin polymerization and myosin-II have distinct roles in phagocytic force generation and progression. a, Confocal images of drug-treated fixed RAW cells phagocytosing DAAM-particles functionalized with AF488-Cadaverine, BSA and anti-BSA IgG. Cells were treated with DMSO, CK666 (150 μM), Blebbistatin (15 μM) and SMIFH2 (10 μM) for 30 minutes prior to phagocytic challenge. Each target is approximately 60% engulfed. Fixed cells were stained for F-actin, and particles were labelled with a fluorescent secondary antibody to reveal the exposed surface. Left column: composite maximum intensity projections (MIP) of confocal z-stacks, 2 nd -3 rd column: single confocal slices through particle centroid. Scale bar, 5μm. b, Particle shape reconstructions from a, revealing detailed target deformations and localization of F-actin over the particle surface. Stars mark the base of the phagocytic cup, and the phagocytic axis is horizontal. Scale bars, 3 μm. c, Normal and shear stresses exerted on the target. Negative normal forces denote (inward) compressive forces. d, Average deformation and F-actin intensity profiles along the phagocytic axis to the cup rim. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets in 30 bins. Targets before 40% engulfment were excluded. e, f, Violin plots of all measured particles, showing individual phagocytic events (colored markers), mean (black cross) and median (dashed line). e, F-actin peak intensity and width. f, F-actin intensity in the cup (behind the rim), measured right (3μm) behind the main peak for each particle. g, Upper panel, cumulative distribution function of the engulfment stage of randomly selected phagocytic events (n = 68, 63, 73 55 respectively) from 3 independent experiments. Two sample Kolmogorov-Smirnov test was used (p = 0.016*). Lower panel, fraction late-stage cups. Error bars indicate st.d. estimated by treating phagocytosis as a Bernoulli process. Fisher’s exact test was used to compare fractions (p = 1.9×10 −4 )***. h, Sphericity and i, constriction magnitude of DAAM particle changes with phagocytic progression upon drug treatment. Colored markers indicate individual events, black lines indicate averages within 5 bins. Right column, violin plots of all events. Marker and line styles as in e. All statistical tests were two-side Wilcoxon rank sum test comparing with the DMSO control (gray) over the same bin with significance levels: p

    Article Snippet: Phagocytosis assay DAAM particles were washed 3X in sterile PBS and opsonized with rabbit anti-BSA antibody (MP Biomedicals, 0865111) for 1 h at room temperature.

    Techniques: Staining, Marker

    Phagocytic forces include strong actin-mediated constriction and increase with phagocytic progression. a, Confocal images of fixed RAW macrophages phagocytosing DAAM-particles functionalized with AF488-Cadaverine, BSA and anti-BSA IgG. Cells were stained for F-actin, and particles with a fluorescent secondary antibody to reveal the exposed surface. Left column: composite maximum intensity projections (MIP) of confocal z-stacks, 2 nd to 4 th column: single confocal slices through particle centroid. Scale bar, 5 μm. b, 3D shape reconstructions of particle in a revealing detailed target deformations and localization of F-actin over the particle surface. Scale bars are 3 μm. Stars mark the base of the phagocytic cup, and the phagocytic axis is horizontal (see e ). c, Normal and shear stresses exerted on the targets in a,b . Negative normal forces denote (inward) compressive forces. d, Averages of absolute magnitudes of compressive and shear stresses for 18 phagocytic cups spread over various stages of engulfment. Violin plots show individual phagocytic events (blue markers), mean (black cross) and median (dashed line). *Two-side Wilcoxon rank sum test: p = 2.0×10 −4 . e, Schematic representation of phagocytic parametrization. Normalized cup position indicates the position along the phagocytic axis relative to the rim of the cup, with 0 the cup base and 1 the rim of the phagocytic cup regardless of engulfment stage. f, Average deformation and F-actin intensity profiles along the phagocytic axis to the cup rim. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets in 30 bins. Targets beyond 40% engulfment were included (54 out of 68 events in total). g, Cumulative distribution function of the engulfment stage of randomly selected phagocytic events (n = 68). Dashed red line indicates a linear fit. h, Target sphericity and elongation depend on phagocytic stage. Blue squares indicate individual measurements, black lines indicate averages within 5 bins. Middle graph inset schematic shows how relative elongation was determined. i, Analysis of particle deformation and F-actin fluorescence along the phagocytic axis for all phagocytic events (n = 68). Marker and line styles as in h . All error bars indicate s.e.m. unless indicated otherwise. j, Analysis of forces in the contractile ring at the cup rim and throughout the remainder of the cup for 18 cups selected for force analysis.

    Journal: bioRxiv

    Article Title: Phagocytic “teeth” and myosin-II “jaw” power target constriction during phagocytosis

    doi: 10.1101/2021.03.14.435346

    Figure Lengend Snippet: Phagocytic forces include strong actin-mediated constriction and increase with phagocytic progression. a, Confocal images of fixed RAW macrophages phagocytosing DAAM-particles functionalized with AF488-Cadaverine, BSA and anti-BSA IgG. Cells were stained for F-actin, and particles with a fluorescent secondary antibody to reveal the exposed surface. Left column: composite maximum intensity projections (MIP) of confocal z-stacks, 2 nd to 4 th column: single confocal slices through particle centroid. Scale bar, 5 μm. b, 3D shape reconstructions of particle in a revealing detailed target deformations and localization of F-actin over the particle surface. Scale bars are 3 μm. Stars mark the base of the phagocytic cup, and the phagocytic axis is horizontal (see e ). c, Normal and shear stresses exerted on the targets in a,b . Negative normal forces denote (inward) compressive forces. d, Averages of absolute magnitudes of compressive and shear stresses for 18 phagocytic cups spread over various stages of engulfment. Violin plots show individual phagocytic events (blue markers), mean (black cross) and median (dashed line). *Two-side Wilcoxon rank sum test: p = 2.0×10 −4 . e, Schematic representation of phagocytic parametrization. Normalized cup position indicates the position along the phagocytic axis relative to the rim of the cup, with 0 the cup base and 1 the rim of the phagocytic cup regardless of engulfment stage. f, Average deformation and F-actin intensity profiles along the phagocytic axis to the cup rim. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets in 30 bins. Targets beyond 40% engulfment were included (54 out of 68 events in total). g, Cumulative distribution function of the engulfment stage of randomly selected phagocytic events (n = 68). Dashed red line indicates a linear fit. h, Target sphericity and elongation depend on phagocytic stage. Blue squares indicate individual measurements, black lines indicate averages within 5 bins. Middle graph inset schematic shows how relative elongation was determined. i, Analysis of particle deformation and F-actin fluorescence along the phagocytic axis for all phagocytic events (n = 68). Marker and line styles as in h . All error bars indicate s.e.m. unless indicated otherwise. j, Analysis of forces in the contractile ring at the cup rim and throughout the remainder of the cup for 18 cups selected for force analysis.

    Article Snippet: Phagocytosis assay DAAM particles were washed 3X in sterile PBS and opsonized with rabbit anti-BSA antibody (MP Biomedicals, 0865111) for 1 h at room temperature.

    Techniques: Staining, Fluorescence, Marker

    Multiple actin regulatory proteins localize to phagocytic teeth. a, RAW macrophages were transfected with fluorescently tagged actin binding proteins and challenged to ingest DAAMPs (11 μm, 1.4 kPa) functionalized with AF647-Cadaverine, BSA and anti-BSA IgG to assess localization to actin teeth (yellow arrowheads). Images are maximum intensity projections of confocal Z-stacks. White boxes in leftmost panels indicate the site of the zoomed images to the right. Scale bar, 5 μm. Zoom scale bar, 1 μm. b, DAAM-particle reconstructions for examples shown in a, showing target deformations and localization of fluorescent proteins with respect to actin teeth. Scale bar, 3 μm. c, Myosin-II condensing into thick concentric rings (marked by orange arrowheads) during late-stage phagocytosis of a highly deformed target. Images are maximum intensity projections of confocal Z-stacks. Scale bar, 5 μm. Zoom scale bar, 1 μm.

    Journal: bioRxiv

    Article Title: Phagocytic “teeth” and myosin-II “jaw” power target constriction during phagocytosis

    doi: 10.1101/2021.03.14.435346

    Figure Lengend Snippet: Multiple actin regulatory proteins localize to phagocytic teeth. a, RAW macrophages were transfected with fluorescently tagged actin binding proteins and challenged to ingest DAAMPs (11 μm, 1.4 kPa) functionalized with AF647-Cadaverine, BSA and anti-BSA IgG to assess localization to actin teeth (yellow arrowheads). Images are maximum intensity projections of confocal Z-stacks. White boxes in leftmost panels indicate the site of the zoomed images to the right. Scale bar, 5 μm. Zoom scale bar, 1 μm. b, DAAM-particle reconstructions for examples shown in a, showing target deformations and localization of fluorescent proteins with respect to actin teeth. Scale bar, 3 μm. c, Myosin-II condensing into thick concentric rings (marked by orange arrowheads) during late-stage phagocytosis of a highly deformed target. Images are maximum intensity projections of confocal Z-stacks. Scale bar, 5 μm. Zoom scale bar, 1 μm.

    Article Snippet: Phagocytosis assay DAAM particles were washed 3X in sterile PBS and opsonized with rabbit anti-BSA antibody (MP Biomedicals, 0865111) for 1 h at room temperature.

    Techniques: Transfection, Binding Assay

    Combined MP-TFM and LLSM reveals phagocytic force-induced deformations in real time. RAW macrophages transfected with mEmerald-Lifeact were fed DAAM-particles (9μm,1.4 kPa) functionalized with AF647-Cadaverine, BSA and anti-BSA IgG and imaged using lattice light-sheet microscopy (LLSM). a, Time lapse montage (min:s) of maximum intensity projections in x/y and x/z. Scale bar, 5 μm b,c Schematic of the combined LLSM and MP-TFM experimental approach and analysis, respectively. d, Front and side view of reconstructed DAAM-particle internalized in a showing target deformations and F-actin localization on particle surface. Colorscale represents the deviation of each vertex from a perfect sphere with radius equal to the median radial distance of all edge coordinates to the particle centroid. Scale bar, 3 μm.

    Journal: bioRxiv

    Article Title: Phagocytic “teeth” and myosin-II “jaw” power target constriction during phagocytosis

    doi: 10.1101/2021.03.14.435346

    Figure Lengend Snippet: Combined MP-TFM and LLSM reveals phagocytic force-induced deformations in real time. RAW macrophages transfected with mEmerald-Lifeact were fed DAAM-particles (9μm,1.4 kPa) functionalized with AF647-Cadaverine, BSA and anti-BSA IgG and imaged using lattice light-sheet microscopy (LLSM). a, Time lapse montage (min:s) of maximum intensity projections in x/y and x/z. Scale bar, 5 μm b,c Schematic of the combined LLSM and MP-TFM experimental approach and analysis, respectively. d, Front and side view of reconstructed DAAM-particle internalized in a showing target deformations and F-actin localization on particle surface. Colorscale represents the deviation of each vertex from a perfect sphere with radius equal to the median radial distance of all edge coordinates to the particle centroid. Scale bar, 3 μm.

    Article Snippet: Phagocytosis assay DAAM particles were washed 3X in sterile PBS and opsonized with rabbit anti-BSA antibody (MP Biomedicals, 0865111) for 1 h at room temperature.

    Techniques: Transfection, Microscopy

    Proximal localization of Shot and its mediation of T antigen trafficking specifically to the proximal segment. ( a – a” ) Localization of T antigens, Shot, and HRP antigens in a permeabilized wild-type primary cultured neuron. Similar to T antigens, Shot was localized in the proximal axon segment in 40% of the permeabilized neurons ( a’ ). The intra-axonal boundary (filled arrowheads) defined by localization of Shot was consistent with that defined by T antigens. ( b ) Fluorescence intensity of T antigens and Shot along axons. The filled arrowhead indicates the intra-axonal boundary. ( c – c” ) Localization of T antigens, ROBO3, and HRP antigens on the surface of a wild-type neuron. T antigens are localized on the surface of the proximal axon segment. Conversely, ROBO3 is localized on the surface of the distal axon segment. Filled and open arrowheads indicate the intra-axonal boundaries defined by the localization of T antigens and ROBO3, respectively ( c , d ). ( d ) Fluorescence intensity of T antigens, ROBO3, and HRP antigens along axons. ( e – e” ) Localization of T antigens, Shot, and HRP antigens in a wild-type embryo at stage 16. T antigens and Shot were both expressed in the CNS and in the PNS. ( f , f’ ) Localization of T and HRP antigens on the surface of a shot 3 / shot SF20 transheterozygous mutant neuron. ( g , g’ ) Localization of T and HRP antigens in a shot 3 / shot SF20 mutant permeabilized neuron. ( h ) Histogram showing the localization of T antigens in/on the wild-type and shot 3 / shot SF20 mutant neurons. The histogram of the surface expression for the wild-type is the same as that at 24 h (shown in Fig. 1h ). In the shot 3 / shot SF20 mutant, the percentage of neurons with proximal localization of T antigens (green) was significantly decreased. P -values were calculated using the G-test. ** p = 3.3 × 10 −14 , * p = 1.8 × 10 −12 . Scale bar: 10 μm ( a , c , f , g ), 50 μm ( e ).

    Journal: Scientific Reports

    Article Title: Short stop mediates axonal compartmentalization of mucin-type core 1 glycans

    doi: 10.1038/srep41455

    Figure Lengend Snippet: Proximal localization of Shot and its mediation of T antigen trafficking specifically to the proximal segment. ( a – a” ) Localization of T antigens, Shot, and HRP antigens in a permeabilized wild-type primary cultured neuron. Similar to T antigens, Shot was localized in the proximal axon segment in 40% of the permeabilized neurons ( a’ ). The intra-axonal boundary (filled arrowheads) defined by localization of Shot was consistent with that defined by T antigens. ( b ) Fluorescence intensity of T antigens and Shot along axons. The filled arrowhead indicates the intra-axonal boundary. ( c – c” ) Localization of T antigens, ROBO3, and HRP antigens on the surface of a wild-type neuron. T antigens are localized on the surface of the proximal axon segment. Conversely, ROBO3 is localized on the surface of the distal axon segment. Filled and open arrowheads indicate the intra-axonal boundaries defined by the localization of T antigens and ROBO3, respectively ( c , d ). ( d ) Fluorescence intensity of T antigens, ROBO3, and HRP antigens along axons. ( e – e” ) Localization of T antigens, Shot, and HRP antigens in a wild-type embryo at stage 16. T antigens and Shot were both expressed in the CNS and in the PNS. ( f , f’ ) Localization of T and HRP antigens on the surface of a shot 3 / shot SF20 transheterozygous mutant neuron. ( g , g’ ) Localization of T and HRP antigens in a shot 3 / shot SF20 mutant permeabilized neuron. ( h ) Histogram showing the localization of T antigens in/on the wild-type and shot 3 / shot SF20 mutant neurons. The histogram of the surface expression for the wild-type is the same as that at 24 h (shown in Fig. 1h ). In the shot 3 / shot SF20 mutant, the percentage of neurons with proximal localization of T antigens (green) was significantly decreased. P -values were calculated using the G-test. ** p = 3.3 × 10 −14 , * p = 1.8 × 10 −12 . Scale bar: 10 μm ( a , c , f , g ), 50 μm ( e ).

    Article Snippet: The following primary antibodies were used: rabbit anti-HRP (1:1000; MP Biomedicals), mouse anti-Robo3 extracellular (1:10; Developmental Studies Hybridoma Bank [DSHB]), mouse anti-Shot (1:10; DSHB), mouse BP102 anti-CNS axons (1:10; DSHB), rabbit anti-α-tubulin (1:200; MBL), and mouse anti-α-tubulin antibodies (1:200; Sigma).

    Techniques: Cell Culture, Fluorescence, Mutagenesis, Expressing