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    Rabbit Immunoglobulin G Protein G
    Description:
    This is a GIBCO Custom Product Please complete and submit the Custom Media Inquiry Form and a Customer Service Representative will contact you regarding pricing and availability
    Catalog Number:
    30029011
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    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher rabbit igg
    Inhibition of ERK1/2 activation impairs PDCoV propagation. (A) ST cells were preincubated with DMSO, PD98059 (50 and 100 μM), or U0126 (50 and 100 μM) for 1 h prior to infection and were mock infected or infected with PDCoV at an MOI of 1. The virus-infected cells were further maintained for 12 h in the presence of DMSO or inhibitors. PDCoV-specific CPEs were monitored and photographed at 12 hpi under an inverted microscope at the magnification of 200× (first panels). For immunostaining, the infected cells were fixed at 12 hpi and incubated with MAb against the PDCoV N protein, followed by incubation with <t>Alexa</t> green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined under a fluorescent microscope at 200× magnification. (B) PDCoV production in the presence of each inhibitor was quantified by measuring the percentage of cells expressing N proteins through flow cytometry. (C) Chemical inhibition of ERK1/2 was quantitatively determined using a FACE assay. ST cells were mock infected or PDCoV infected in the presence of DMSO, PD98059, or U0126. The cells were fixed at 6 hpi with 4% formaldehyde and incubated with an anti-ERK1/2 or anti-phospho-ERK1/2 antibody followed by HRP-conjugated <t>IgG</t> antibodies. The absorbance of the solution was determined at 450 nm using a spectrophotometer. (D) At 6 hpi, cellular lysates were prepared and subjected to immunoblotting using an antibody against p-ERK1/2, ERK1/2, p-Elk-1, or PDCoV N. The blot was also reacted with an anti-β-actin antibody to verify equal protein leading. Each protein expression was quantitatively analyzed by densitometry, and fold changes in each p-ERK1/2:total ERK1/2, p-Elk-1:β-actin, and PDCoV N:β-actin ratio are independently plotted (right panel). Data are the representative of the means from three independent experiments, and error bars denote the mean ± SDM. * P
    This is a GIBCO Custom Product Please complete and submit the Custom Media Inquiry Form and a Customer Service Representative will contact you regarding pricing and availability
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    Images

    1) Product Images from "Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication"

    Article Title: Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication

    Journal: Virus Research

    doi: 10.1016/j.virusres.2020.197961

    Inhibition of ERK1/2 activation impairs PDCoV propagation. (A) ST cells were preincubated with DMSO, PD98059 (50 and 100 μM), or U0126 (50 and 100 μM) for 1 h prior to infection and were mock infected or infected with PDCoV at an MOI of 1. The virus-infected cells were further maintained for 12 h in the presence of DMSO or inhibitors. PDCoV-specific CPEs were monitored and photographed at 12 hpi under an inverted microscope at the magnification of 200× (first panels). For immunostaining, the infected cells were fixed at 12 hpi and incubated with MAb against the PDCoV N protein, followed by incubation with Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined under a fluorescent microscope at 200× magnification. (B) PDCoV production in the presence of each inhibitor was quantified by measuring the percentage of cells expressing N proteins through flow cytometry. (C) Chemical inhibition of ERK1/2 was quantitatively determined using a FACE assay. ST cells were mock infected or PDCoV infected in the presence of DMSO, PD98059, or U0126. The cells were fixed at 6 hpi with 4% formaldehyde and incubated with an anti-ERK1/2 or anti-phospho-ERK1/2 antibody followed by HRP-conjugated IgG antibodies. The absorbance of the solution was determined at 450 nm using a spectrophotometer. (D) At 6 hpi, cellular lysates were prepared and subjected to immunoblotting using an antibody against p-ERK1/2, ERK1/2, p-Elk-1, or PDCoV N. The blot was also reacted with an anti-β-actin antibody to verify equal protein leading. Each protein expression was quantitatively analyzed by densitometry, and fold changes in each p-ERK1/2:total ERK1/2, p-Elk-1:β-actin, and PDCoV N:β-actin ratio are independently plotted (right panel). Data are the representative of the means from three independent experiments, and error bars denote the mean ± SDM. * P
    Figure Legend Snippet: Inhibition of ERK1/2 activation impairs PDCoV propagation. (A) ST cells were preincubated with DMSO, PD98059 (50 and 100 μM), or U0126 (50 and 100 μM) for 1 h prior to infection and were mock infected or infected with PDCoV at an MOI of 1. The virus-infected cells were further maintained for 12 h in the presence of DMSO or inhibitors. PDCoV-specific CPEs were monitored and photographed at 12 hpi under an inverted microscope at the magnification of 200× (first panels). For immunostaining, the infected cells were fixed at 12 hpi and incubated with MAb against the PDCoV N protein, followed by incubation with Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined under a fluorescent microscope at 200× magnification. (B) PDCoV production in the presence of each inhibitor was quantified by measuring the percentage of cells expressing N proteins through flow cytometry. (C) Chemical inhibition of ERK1/2 was quantitatively determined using a FACE assay. ST cells were mock infected or PDCoV infected in the presence of DMSO, PD98059, or U0126. The cells were fixed at 6 hpi with 4% formaldehyde and incubated with an anti-ERK1/2 or anti-phospho-ERK1/2 antibody followed by HRP-conjugated IgG antibodies. The absorbance of the solution was determined at 450 nm using a spectrophotometer. (D) At 6 hpi, cellular lysates were prepared and subjected to immunoblotting using an antibody against p-ERK1/2, ERK1/2, p-Elk-1, or PDCoV N. The blot was also reacted with an anti-β-actin antibody to verify equal protein leading. Each protein expression was quantitatively analyzed by densitometry, and fold changes in each p-ERK1/2:total ERK1/2, p-Elk-1:β-actin, and PDCoV N:β-actin ratio are independently plotted (right panel). Data are the representative of the means from three independent experiments, and error bars denote the mean ± SDM. * P

    Techniques Used: Inhibition, Activation Assay, Infection, Inverted Microscopy, Immunostaining, Incubation, Microscopy, Expressing, Flow Cytometry, Spectrophotometry

    2) Product Images from "A Novel Monoclonal Antibody Targets Mucin1 and Attenuates Growth in Pancreatic Cancer Model"

    Article Title: A Novel Monoclonal Antibody Targets Mucin1 and Attenuates Growth in Pancreatic Cancer Model

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19072004

    Time-dependent internalization of anti-hMUC1 monoclonal antibody into pancreatic cancer cells. Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were incubated with DyLight 488-conjugated anti-hMUC1 monoclonal antibody for various time intervals and analyzed by confocal microscopy. Hoechst 33258 was used for staining nuclei. Scale bars, 10 µm. These results are representatives of three independent experiments.
    Figure Legend Snippet: Time-dependent internalization of anti-hMUC1 monoclonal antibody into pancreatic cancer cells. Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were incubated with DyLight 488-conjugated anti-hMUC1 monoclonal antibody for various time intervals and analyzed by confocal microscopy. Hoechst 33258 was used for staining nuclei. Scale bars, 10 µm. These results are representatives of three independent experiments.

    Techniques Used: Incubation, Confocal Microscopy, Staining

    Biodistribution of anti-hMUC1 monoclonal antibody in a xenograft mouse model. BALB/c nu/nu mice were subcutaneously inoculated with Capan-2 cancer cells to allow tumor formation. ( a ) DyLight 755-labeled normal IgG (5 mg/kg) or DyLight 755-labeled anti-hMUC1 monoclonal antibody (5 mg/kg) were intravenously injected into mice, followed by acquisition of whole body fluorescent images at 0, 24 and 48 h using in vivo imaging system. ( b ) Upper panel: The localization of the antibody in the tumor region intact to mice. Lower panel: The localization of the antibody in the tumor region after dissection from the same mice. ( c ) Tumor tissues and organs were isolated and analyzed for the antibody distribution. Scale bars, 2.5 cm ( a – c ). ( d ) Confocal images of tumor sections obtained from mice intravenously injected with DyLight 488-labeled normal IgG or anti-hMUC1 monoclonal antibody. The frozen tissues were cut into slices using a cryostat and stained with Hoechst 33258 for nuclei. The mounted samples were examined with confocal microscopy. Scale bars, 10 µm ( d ). The images are representative of data from 3 sets of mice.
    Figure Legend Snippet: Biodistribution of anti-hMUC1 monoclonal antibody in a xenograft mouse model. BALB/c nu/nu mice were subcutaneously inoculated with Capan-2 cancer cells to allow tumor formation. ( a ) DyLight 755-labeled normal IgG (5 mg/kg) or DyLight 755-labeled anti-hMUC1 monoclonal antibody (5 mg/kg) were intravenously injected into mice, followed by acquisition of whole body fluorescent images at 0, 24 and 48 h using in vivo imaging system. ( b ) Upper panel: The localization of the antibody in the tumor region intact to mice. Lower panel: The localization of the antibody in the tumor region after dissection from the same mice. ( c ) Tumor tissues and organs were isolated and analyzed for the antibody distribution. Scale bars, 2.5 cm ( a – c ). ( d ) Confocal images of tumor sections obtained from mice intravenously injected with DyLight 488-labeled normal IgG or anti-hMUC1 monoclonal antibody. The frozen tissues were cut into slices using a cryostat and stained with Hoechst 33258 for nuclei. The mounted samples were examined with confocal microscopy. Scale bars, 10 µm ( d ). The images are representative of data from 3 sets of mice.

    Techniques Used: Mouse Assay, Labeling, Injection, In Vivo Imaging, Dissection, Isolation, Staining, Confocal Microscopy

    Immunoreactivity of anti-hMUC1 monoclonal antibody in pancreatic cancer cells. ( a ) Lysates from the pancreatic cancer cell lines Capan-1, Capan-2, CFPAC-1 and PANC-1 were analyzed by western blotting using anti-MUC1-CT, anti-hMUC1 and anti-β-actin antibodies. ( b ) Capan-1, Capan-2, CFPAC-1 and PANC-1 cell lysates were immunoprecipitated with mouse normal IgG or anti-hMUC1 monoclonal antibody followed by western blotting using anti-MUC1-CT antibody. ( c ) Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were fixed with 4% paraformaldehyde and incubated with mouse normal IgG or anti-hMUC1 monoclonal antibody on ice to identify MUC1-C on the membrane surface of the cells (surface). On the other hand, to identify MUC1-C in the intracellular region, the cells were fixed, permeabilized and incubated with mouse normal IgG or anti-hMUC1 monoclonal antibody at room temperature (permeabilization). Then, the cells were stained with Alexa 488-conjugated secondary antibody. Hoechst 33258 was used for staining nuclei. Images were captured using confocal microscopy. Scale bars, 10 µm. These results are representatives of three independent experiments.
    Figure Legend Snippet: Immunoreactivity of anti-hMUC1 monoclonal antibody in pancreatic cancer cells. ( a ) Lysates from the pancreatic cancer cell lines Capan-1, Capan-2, CFPAC-1 and PANC-1 were analyzed by western blotting using anti-MUC1-CT, anti-hMUC1 and anti-β-actin antibodies. ( b ) Capan-1, Capan-2, CFPAC-1 and PANC-1 cell lysates were immunoprecipitated with mouse normal IgG or anti-hMUC1 monoclonal antibody followed by western blotting using anti-MUC1-CT antibody. ( c ) Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were fixed with 4% paraformaldehyde and incubated with mouse normal IgG or anti-hMUC1 monoclonal antibody on ice to identify MUC1-C on the membrane surface of the cells (surface). On the other hand, to identify MUC1-C in the intracellular region, the cells were fixed, permeabilized and incubated with mouse normal IgG or anti-hMUC1 monoclonal antibody at room temperature (permeabilization). Then, the cells were stained with Alexa 488-conjugated secondary antibody. Hoechst 33258 was used for staining nuclei. Images were captured using confocal microscopy. Scale bars, 10 µm. These results are representatives of three independent experiments.

    Techniques Used: Western Blot, Immunoprecipitation, Incubation, Staining, Confocal Microscopy

    3) Product Images from "Myeloid-Specific Blockade of Notch Signaling Attenuates Choroidal Neovascularization through Compromised Macrophage Infiltration and Polarization in Mice"

    Article Title: Myeloid-Specific Blockade of Notch Signaling Attenuates Choroidal Neovascularization through Compromised Macrophage Infiltration and Polarization in Mice

    Journal: Scientific Reports

    doi: 10.1038/srep28617

    Notch activation in the choroidal tissues of mice with induced CNV. ( A ) The eyes of wild-type C57/B16 mice were subjected to laser-induced CNV. Eye cups were collected at the indicated time points after laser treatment, and were stained with anti-F4/80 and anti-NICD. Arrows indicated NICD + macrophages. ( B ) The two eyes of one mouse were used as one set of RNA retraction. The relative mRNA level of Hes1 and Hey1 was determined by using qRT-PCR, with β-actin as an internal reference control. Each individual experiment was repeated at least three times. Data are presented as mean ± SEM (n = five mice per group). *P
    Figure Legend Snippet: Notch activation in the choroidal tissues of mice with induced CNV. ( A ) The eyes of wild-type C57/B16 mice were subjected to laser-induced CNV. Eye cups were collected at the indicated time points after laser treatment, and were stained with anti-F4/80 and anti-NICD. Arrows indicated NICD + macrophages. ( B ) The two eyes of one mouse were used as one set of RNA retraction. The relative mRNA level of Hes1 and Hey1 was determined by using qRT-PCR, with β-actin as an internal reference control. Each individual experiment was repeated at least three times. Data are presented as mean ± SEM (n = five mice per group). *P

    Techniques Used: Activation Assay, Mouse Assay, Staining, Quantitative RT-PCR

    Myeloid specific RBP-J deficiency decreased macrophage infiltration and M2 macrophage polarization in CNV lesions. ( A ) RBP-J cKO and control mice were subjected to laser coagulation. Choroidal tissues were flat-mounted and stained with anti-F4/80 as indicated time points. Macrophage infiltration area was represented as pixels and the average pixels per CNV lesion were calculated. Histogram shows the comparisons on the average area of macrophage infiltration in five mice per group. ( B ) Two eyes of one mouse were adopted as one set to prepare the single cell suspensions from RPE-choroidal tissues in ( A ) at day 3 after laser injury, and analyzed by flow cytometry. The numbers of F4/80 + CD11b + cells were compared between the two groups (five mice per group). ( C ) Retinal tissues of mice in ( A ) were immunolabeled with F4/80 and Arg1 or F4/80 and iNOS at day 3 after injury. Arrow indicates Arg1 + macrophages or iNOS + macrophages. Three representative images per lesion were randomly selected from three biggest CNV lesions in each eye for cell count, and the average number of F4/80 + Arg1 + or F4/80 + iNOS + macrophages was calculated and compared in five eyes per group. ( D ) Flat-mounted choroidal tissues of mice in ( C ) were immunolabeled with Arg1 and iNOS. The total pixels of iNOS and Arg1 were measured to calculate the ratio of M1/M2. The ratio of M1/M2 was compared between two groups (five eyes per group). ( E ) Total RNA was prepared from choroidal lysates of RBP-J cKO and control mice at day 3 after laser injury. Two eyes of one mouse were adopted to prepare the RNA retraction for qPCR. The mRNA level of Arg1, iNOS, and IL-6 determined with qRT-PCR, with β-actin as an internal reference control. Each individual experiment was repeated at least three times. The expressions of Arg1, iNOS, IL-6, and the ratio of iNOS/Arg1 were compared. Data are presented as mean ± SEM of five eyes or mice per group. *P
    Figure Legend Snippet: Myeloid specific RBP-J deficiency decreased macrophage infiltration and M2 macrophage polarization in CNV lesions. ( A ) RBP-J cKO and control mice were subjected to laser coagulation. Choroidal tissues were flat-mounted and stained with anti-F4/80 as indicated time points. Macrophage infiltration area was represented as pixels and the average pixels per CNV lesion were calculated. Histogram shows the comparisons on the average area of macrophage infiltration in five mice per group. ( B ) Two eyes of one mouse were adopted as one set to prepare the single cell suspensions from RPE-choroidal tissues in ( A ) at day 3 after laser injury, and analyzed by flow cytometry. The numbers of F4/80 + CD11b + cells were compared between the two groups (five mice per group). ( C ) Retinal tissues of mice in ( A ) were immunolabeled with F4/80 and Arg1 or F4/80 and iNOS at day 3 after injury. Arrow indicates Arg1 + macrophages or iNOS + macrophages. Three representative images per lesion were randomly selected from three biggest CNV lesions in each eye for cell count, and the average number of F4/80 + Arg1 + or F4/80 + iNOS + macrophages was calculated and compared in five eyes per group. ( D ) Flat-mounted choroidal tissues of mice in ( C ) were immunolabeled with Arg1 and iNOS. The total pixels of iNOS and Arg1 were measured to calculate the ratio of M1/M2. The ratio of M1/M2 was compared between two groups (five eyes per group). ( E ) Total RNA was prepared from choroidal lysates of RBP-J cKO and control mice at day 3 after laser injury. Two eyes of one mouse were adopted to prepare the RNA retraction for qPCR. The mRNA level of Arg1, iNOS, and IL-6 determined with qRT-PCR, with β-actin as an internal reference control. Each individual experiment was repeated at least three times. The expressions of Arg1, iNOS, IL-6, and the ratio of iNOS/Arg1 were compared. Data are presented as mean ± SEM of five eyes or mice per group. *P

    Techniques Used: Mouse Assay, Coagulation, Staining, Flow Cytometry, Cytometry, Immunolabeling, Cell Counting, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Myeloid VEGF and TNF-α expressions were reduced in CNV lesions of RBP-J cKO mice. ( A ) RBP-J cKO and control mice were subjected to laser coagulation. Choroidal tissues were flat mounted on day 3 and stained with anti-F4/80 and anti-VEGF, and observed under a confocal microscope. The average areas of VEGF expression in 24 randomly selected images were compared between control and RBP-J cKO group. ( B ) Total RNA of two eyes from one mouse was prepared from choroid tissue in ( A ). VEGF expression was determined by using qRT-PCR and compared between control and RBP-J cKO group. ( C ) CNV lesions of mice in ( A ) were sectioned and stained with anti-F4/80 and anti-VEGF, and observed under a confocal microscope. Five representative images from one eye were randomly selected and F4/80 + VEGF + cells were counted and compared in two groups (five eyes from five mice per group). ( D ) Flat mounted choroidal tissues in ( A ) were stained with anti-F4/80 and anti-TNF-α and observed under a confocal microscope. TNF-α immuno-reactivities were compared between control and RBP-J cKO group. ( E ) Total RNA of two eyes from one mouse was prepared from choroid tissue in ( A ). TNF-α expression was determined by using qRT-PCR and compared between control and RBP-J cKO group. ( F ) CNV lesions of mice in ( A ) were sectioned and stained with anti-F4/80 and anti-TNF-α, and observed under a confocal microscope. F4/80 + TNF-α + cells were compared. Five representative images from one eye were randomly selected and F4/80 + TNF-α + cells were counted and compared in two groups (five eyes from five mice per group). Data are presented as mean ± SEM (n = five mice per group). *P
    Figure Legend Snippet: Myeloid VEGF and TNF-α expressions were reduced in CNV lesions of RBP-J cKO mice. ( A ) RBP-J cKO and control mice were subjected to laser coagulation. Choroidal tissues were flat mounted on day 3 and stained with anti-F4/80 and anti-VEGF, and observed under a confocal microscope. The average areas of VEGF expression in 24 randomly selected images were compared between control and RBP-J cKO group. ( B ) Total RNA of two eyes from one mouse was prepared from choroid tissue in ( A ). VEGF expression was determined by using qRT-PCR and compared between control and RBP-J cKO group. ( C ) CNV lesions of mice in ( A ) were sectioned and stained with anti-F4/80 and anti-VEGF, and observed under a confocal microscope. Five representative images from one eye were randomly selected and F4/80 + VEGF + cells were counted and compared in two groups (five eyes from five mice per group). ( D ) Flat mounted choroidal tissues in ( A ) were stained with anti-F4/80 and anti-TNF-α and observed under a confocal microscope. TNF-α immuno-reactivities were compared between control and RBP-J cKO group. ( E ) Total RNA of two eyes from one mouse was prepared from choroid tissue in ( A ). TNF-α expression was determined by using qRT-PCR and compared between control and RBP-J cKO group. ( F ) CNV lesions of mice in ( A ) were sectioned and stained with anti-F4/80 and anti-TNF-α, and observed under a confocal microscope. F4/80 + TNF-α + cells were compared. Five representative images from one eye were randomly selected and F4/80 + TNF-α + cells were counted and compared in two groups (five eyes from five mice per group). Data are presented as mean ± SEM (n = five mice per group). *P

    Techniques Used: Mouse Assay, Coagulation, Staining, Microscopy, Expressing, Quantitative RT-PCR

    4) Product Images from "Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis"

    Article Title: Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139357

    S100A13, but not podoplanin, was expressed on the surface of atherosclerotic lesions. A) Serial frozen-thawed sections of the mouse abdominal aorta from wild type (i) or ApoE- deficient mice fed with high fat diet (ii) were incubated with control rabbit IgG (left panel), anti-smooth muscle actin (middle panel), anti-S100A13 antibody (right panel), followed by anti-rabbit IgG-Alexa Flour 488. Single and double asterisks indicate lumen and atherosclerotic plaque, respectively. B) Serial frozen-thawed sections of the mouse abdominal aorta from ApoE- deficient mice fed with high fat diet were incubated with mCLEC-2-ratFc2 (left panel), anti-S100A13 antibody (middle panel), followed by anti-rat IgG-Alexa Flour 488 or anti-rabbit IgG Alexa Flour 546. Images in left and middle panels were merged (right panel). Lower panels are magnified images of the squared area in the upper panels. C) Serial frozen-thawed sections of the mouse abdominal aorta from ApoE- deficient mice fed with high fat diet were incubated with anti-podoplanin antibody (left panel), anti-S100A13 antibody (middle panel), followed by anti-hamster IgG-Alexa Flour 488 or anti-rabbit IgG Alexa Flour 546. Images in left and middle panels were merged (right panel). All the sections were counter-stained with DAPI.
    Figure Legend Snippet: S100A13, but not podoplanin, was expressed on the surface of atherosclerotic lesions. A) Serial frozen-thawed sections of the mouse abdominal aorta from wild type (i) or ApoE- deficient mice fed with high fat diet (ii) were incubated with control rabbit IgG (left panel), anti-smooth muscle actin (middle panel), anti-S100A13 antibody (right panel), followed by anti-rabbit IgG-Alexa Flour 488. Single and double asterisks indicate lumen and atherosclerotic plaque, respectively. B) Serial frozen-thawed sections of the mouse abdominal aorta from ApoE- deficient mice fed with high fat diet were incubated with mCLEC-2-ratFc2 (left panel), anti-S100A13 antibody (middle panel), followed by anti-rat IgG-Alexa Flour 488 or anti-rabbit IgG Alexa Flour 546. Images in left and middle panels were merged (right panel). Lower panels are magnified images of the squared area in the upper panels. C) Serial frozen-thawed sections of the mouse abdominal aorta from ApoE- deficient mice fed with high fat diet were incubated with anti-podoplanin antibody (left panel), anti-S100A13 antibody (middle panel), followed by anti-hamster IgG-Alexa Flour 488 or anti-rabbit IgG Alexa Flour 546. Images in left and middle panels were merged (right panel). All the sections were counter-stained with DAPI.

    Techniques Used: Mouse Assay, Incubation, Staining

    5) Product Images from "A Novel Monoclonal Antibody Targets Mucin1 and Attenuates Growth in Pancreatic Cancer Model"

    Article Title: A Novel Monoclonal Antibody Targets Mucin1 and Attenuates Growth in Pancreatic Cancer Model

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19072004

    Time-dependent internalization of anti-hMUC1 monoclonal antibody into pancreatic cancer cells. Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were incubated with DyLight 488-conjugated anti-hMUC1 monoclonal antibody for various time intervals and analyzed by confocal microscopy. Hoechst 33258 was used for staining nuclei. Scale bars, 10 µm. These results are representatives of three independent experiments.
    Figure Legend Snippet: Time-dependent internalization of anti-hMUC1 monoclonal antibody into pancreatic cancer cells. Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were incubated with DyLight 488-conjugated anti-hMUC1 monoclonal antibody for various time intervals and analyzed by confocal microscopy. Hoechst 33258 was used for staining nuclei. Scale bars, 10 µm. These results are representatives of three independent experiments.

    Techniques Used: Incubation, Confocal Microscopy, Staining

    Biodistribution of anti-hMUC1 monoclonal antibody in a xenograft mouse model. BALB/c nu/nu mice were subcutaneously inoculated with Capan-2 cancer cells to allow tumor formation. ( a ) DyLight 755-labeled normal IgG (5 mg/kg) or DyLight 755-labeled anti-hMUC1 monoclonal antibody (5 mg/kg) were intravenously injected into mice, followed by acquisition of whole body fluorescent images at 0, 24 and 48 h using in vivo imaging system. ( b ) Upper panel: The localization of the antibody in the tumor region intact to mice. Lower panel: The localization of the antibody in the tumor region after dissection from the same mice. ( c ) Tumor tissues and organs were isolated and analyzed for the antibody distribution. Scale bars, 2.5 cm ( a – c ). ( d ) Confocal images of tumor sections obtained from mice intravenously injected with DyLight 488-labeled normal IgG or anti-hMUC1 monoclonal antibody. The frozen tissues were cut into slices using a cryostat and stained with Hoechst 33258 for nuclei. The mounted samples were examined with confocal microscopy. Scale bars, 10 µm ( d ). The images are representative of data from 3 sets of mice.
    Figure Legend Snippet: Biodistribution of anti-hMUC1 monoclonal antibody in a xenograft mouse model. BALB/c nu/nu mice were subcutaneously inoculated with Capan-2 cancer cells to allow tumor formation. ( a ) DyLight 755-labeled normal IgG (5 mg/kg) or DyLight 755-labeled anti-hMUC1 monoclonal antibody (5 mg/kg) were intravenously injected into mice, followed by acquisition of whole body fluorescent images at 0, 24 and 48 h using in vivo imaging system. ( b ) Upper panel: The localization of the antibody in the tumor region intact to mice. Lower panel: The localization of the antibody in the tumor region after dissection from the same mice. ( c ) Tumor tissues and organs were isolated and analyzed for the antibody distribution. Scale bars, 2.5 cm ( a – c ). ( d ) Confocal images of tumor sections obtained from mice intravenously injected with DyLight 488-labeled normal IgG or anti-hMUC1 monoclonal antibody. The frozen tissues were cut into slices using a cryostat and stained with Hoechst 33258 for nuclei. The mounted samples were examined with confocal microscopy. Scale bars, 10 µm ( d ). The images are representative of data from 3 sets of mice.

    Techniques Used: Mouse Assay, Labeling, Injection, In Vivo Imaging, Dissection, Isolation, Staining, Confocal Microscopy

    Immunoreactivity of anti-hMUC1 monoclonal antibody in pancreatic cancer cells. ( a ) Lysates from the pancreatic cancer cell lines Capan-1, Capan-2, CFPAC-1 and PANC-1 were analyzed by western blotting using anti-MUC1-CT, anti-hMUC1 and anti-β-actin antibodies. ( b ) Capan-1, Capan-2, CFPAC-1 and PANC-1 cell lysates were immunoprecipitated with mouse normal IgG or anti-hMUC1 monoclonal antibody followed by western blotting using anti-MUC1-CT antibody. ( c ) Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were fixed with 4% paraformaldehyde and incubated with mouse normal IgG or anti-hMUC1 monoclonal antibody on ice to identify MUC1-C on the membrane surface of the cells (surface). On the other hand, to identify MUC1-C in the intracellular region, the cells were fixed, permeabilized and incubated with mouse normal IgG or anti-hMUC1 monoclonal antibody at room temperature (permeabilization). Then, the cells were stained with Alexa 488-conjugated secondary antibody. Hoechst 33258 was used for staining nuclei. Images were captured using confocal microscopy. Scale bars, 10 µm. These results are representatives of three independent experiments.
    Figure Legend Snippet: Immunoreactivity of anti-hMUC1 monoclonal antibody in pancreatic cancer cells. ( a ) Lysates from the pancreatic cancer cell lines Capan-1, Capan-2, CFPAC-1 and PANC-1 were analyzed by western blotting using anti-MUC1-CT, anti-hMUC1 and anti-β-actin antibodies. ( b ) Capan-1, Capan-2, CFPAC-1 and PANC-1 cell lysates were immunoprecipitated with mouse normal IgG or anti-hMUC1 monoclonal antibody followed by western blotting using anti-MUC1-CT antibody. ( c ) Capan-1, Capan-2, CFPAC-1 and PANC-1 cells were fixed with 4% paraformaldehyde and incubated with mouse normal IgG or anti-hMUC1 monoclonal antibody on ice to identify MUC1-C on the membrane surface of the cells (surface). On the other hand, to identify MUC1-C in the intracellular region, the cells were fixed, permeabilized and incubated with mouse normal IgG or anti-hMUC1 monoclonal antibody at room temperature (permeabilization). Then, the cells were stained with Alexa 488-conjugated secondary antibody. Hoechst 33258 was used for staining nuclei. Images were captured using confocal microscopy. Scale bars, 10 µm. These results are representatives of three independent experiments.

    Techniques Used: Western Blot, Immunoprecipitation, Incubation, Staining, Confocal Microscopy

    6) Product Images from "microRNAs (miR 9, 124, 155 and 224) transdifferentiate macrophages to neurons"

    Article Title: microRNAs (miR 9, 124, 155 and 224) transdifferentiate macrophages to neurons

    Journal: bioRxiv

    doi: 10.1101/2020.07.19.210633

    miR-mediated reprogramming involves stem cell-like intermediates. Macrophages were transfected with miRs twice and analysed for Stem cell markers, neuronal marker and pluripotency genes. (A) Flow cytometry analysis of transfected cells were done for NSE and Prominin (A’) NSE expressing cells are represented through histogram (n=3). (B) Relative density of the genes to β -actin were calculated and plotted as Heat Map considering Pu.1 in miR 224 as least and Oct4 in miR 155 as highest expression (C) miR 302 levels were measured RT-PCR in miR transfected cells. (D) Reprogrammed cells were selected on the basis of expression of CD11b and CD11b - cells were further selected for CD133. % of CD11b + ’ CD11b - CD133 + and CD11b - CD133 - cells were calculated to initial cells seeded and plotted. (E) CD133 + cells and CD11b - cells were further transfected once with miRs and stained for neuronal markers Nurr1 and Map2 and observed through immunofluorescence microscope. (400X magnification).
    Figure Legend Snippet: miR-mediated reprogramming involves stem cell-like intermediates. Macrophages were transfected with miRs twice and analysed for Stem cell markers, neuronal marker and pluripotency genes. (A) Flow cytometry analysis of transfected cells were done for NSE and Prominin (A’) NSE expressing cells are represented through histogram (n=3). (B) Relative density of the genes to β -actin were calculated and plotted as Heat Map considering Pu.1 in miR 224 as least and Oct4 in miR 155 as highest expression (C) miR 302 levels were measured RT-PCR in miR transfected cells. (D) Reprogrammed cells were selected on the basis of expression of CD11b and CD11b - cells were further selected for CD133. % of CD11b + ’ CD11b - CD133 + and CD11b - CD133 - cells were calculated to initial cells seeded and plotted. (E) CD133 + cells and CD11b - cells were further transfected once with miRs and stained for neuronal markers Nurr1 and Map2 and observed through immunofluorescence microscope. (400X magnification).

    Techniques Used: Transfection, Marker, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Immunofluorescence, Microscopy

    miR mimics transdifferentiate macrophages to induced Neuronal cells (iNCs). (A) Macrophages were transfected with miRs as indicated earlier for three times and gene expression was analysed by sq-RT PCR for Nestin and CD11b . (B) Immunofluorescence of a myeloid integrin, CD11c of reprogrammed cells. (B’) Cells expressing CD11c were counted in three random fields and plotted as % of CD11c + cells. Real time PCR analysis of Map2 (C), Tubb3 (D) and Mash1 (E) in neuron-like cells. (F and F’) Mature neural marker, NSE and NGFR were analysed by sq-RT PCR. (G and G’) Increase in neural markers NSE and Map2 was analysed using Western blot in iNCs. iNCs were fluorescently stained with various neuronal markers: (H) Map2 and Nurr1; (I) Nestin and NSE; (J) GFAP and (K) N-Cadherin. (400X magnification). (See supplementary figures S3A, S3B, S3C, S3D aslo) (Relative density was plotted using data from three individual experiments).
    Figure Legend Snippet: miR mimics transdifferentiate macrophages to induced Neuronal cells (iNCs). (A) Macrophages were transfected with miRs as indicated earlier for three times and gene expression was analysed by sq-RT PCR for Nestin and CD11b . (B) Immunofluorescence of a myeloid integrin, CD11c of reprogrammed cells. (B’) Cells expressing CD11c were counted in three random fields and plotted as % of CD11c + cells. Real time PCR analysis of Map2 (C), Tubb3 (D) and Mash1 (E) in neuron-like cells. (F and F’) Mature neural marker, NSE and NGFR were analysed by sq-RT PCR. (G and G’) Increase in neural markers NSE and Map2 was analysed using Western blot in iNCs. iNCs were fluorescently stained with various neuronal markers: (H) Map2 and Nurr1; (I) Nestin and NSE; (J) GFAP and (K) N-Cadherin. (400X magnification). (See supplementary figures S3A, S3B, S3C, S3D aslo) (Relative density was plotted using data from three individual experiments).

    Techniques Used: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Real-time Polymerase Chain Reaction, Marker, Western Blot, Staining

    7) Product Images from "α- Linoleanic acid modulates phagocytosis of extracellular Tau and induces microglial migration by actin-remodeling"

    Article Title: α- Linoleanic acid modulates phagocytosis of extracellular Tau and induces microglial migration by actin-remodeling

    Journal: bioRxiv

    doi: 10.1101/2020.04.15.042143

    Extracellular Tau aggregates internalization-induced by α-Linolenic acid in Microglia. Internalization hTau40 recombinant Tau in Iba-1 positive microglia. A) ALA is one of the omega-3 fatty acids which has major role on cell membrane modulation of microglia, increased ALA in diet induces cell membrane changes that enhances the anti-inflammatory phenotype of microglia, we hypothesize that increased phagocytic ability of microglia may clear the extracellular Tau species which are responsible for Tau seeding across the healthy cells. B) Cells were incubated with hTau40 aggregates species, hTau40 monomer species (1μM) alone and along with the α-Linolenic acid (40μM) for 24 hours at 37 degrees as control 40 μM ALA alone and cell control (without treatment) was kept for comparison. The cells were fixed after 24 hours and stained with anti-Iba-1 antibody (green) and T46 Tau antibody (red) and observe by fluorescence microscopy, scale bar is 20μm. The enlarged image showing the zoomed areas of microglia with internalized Tau marked with white arrow marks. C) 3D view of internalized recombinant hTau40 in Iba-1 positive microglia. 3D view helps to show the localization of internalized Tau in microglia. The images were taken with the Zeiss fluorescent microscope with Apotome 2.0. E) Quantification of internalized Tau intensity per unit square area of microglia cells; showing extent of internalization Tau in microglia cells which is highly significant P
    Figure Legend Snippet: Extracellular Tau aggregates internalization-induced by α-Linolenic acid in Microglia. Internalization hTau40 recombinant Tau in Iba-1 positive microglia. A) ALA is one of the omega-3 fatty acids which has major role on cell membrane modulation of microglia, increased ALA in diet induces cell membrane changes that enhances the anti-inflammatory phenotype of microglia, we hypothesize that increased phagocytic ability of microglia may clear the extracellular Tau species which are responsible for Tau seeding across the healthy cells. B) Cells were incubated with hTau40 aggregates species, hTau40 monomer species (1μM) alone and along with the α-Linolenic acid (40μM) for 24 hours at 37 degrees as control 40 μM ALA alone and cell control (without treatment) was kept for comparison. The cells were fixed after 24 hours and stained with anti-Iba-1 antibody (green) and T46 Tau antibody (red) and observe by fluorescence microscopy, scale bar is 20μm. The enlarged image showing the zoomed areas of microglia with internalized Tau marked with white arrow marks. C) 3D view of internalized recombinant hTau40 in Iba-1 positive microglia. 3D view helps to show the localization of internalized Tau in microglia. The images were taken with the Zeiss fluorescent microscope with Apotome 2.0. E) Quantification of internalized Tau intensity per unit square area of microglia cells; showing extent of internalization Tau in microglia cells which is highly significant P

    Techniques Used: Recombinant, Incubation, Staining, Fluorescence, Microscopy

    8) Product Images from "UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage–specific manner"

    Article Title: UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage–specific manner

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201609100

    Effect of deleting BBF2H7 on formation of the perinotochordal basement membrane. (A) Double immunofluorescence analysis of the notochord at stage 27 of WT and BBF2H7- KO medaka using anti-calnexin and anti–type II collagen antibodies as well as DAPI staining. Areas surrounded by the white dashed boxes are enlarged beneath each panel. The dashed line in the bottom right WT panel shows the separation of the sheath cell–containing zone (right) from the vacuolated cell–containing zone (left). (B) Immunofluorescence analysis using the anti–type II collagen antibody along with fluorescence microscopic analysis of an area of the notochord of BBF2H7 -KO medaka carrying P brachyury - Venus at stage 27, where the vacuolization was completed. Bars, 10 µm. (C) Schematic representation of notochord structure after vacuolization. (D) Enlarged area surrounded by the yellow dashed line in A. p, perinotochordal basement membrane; s, sheath cell; v, vacuolated cell. Scale is the same as other insets.
    Figure Legend Snippet: Effect of deleting BBF2H7 on formation of the perinotochordal basement membrane. (A) Double immunofluorescence analysis of the notochord at stage 27 of WT and BBF2H7- KO medaka using anti-calnexin and anti–type II collagen antibodies as well as DAPI staining. Areas surrounded by the white dashed boxes are enlarged beneath each panel. The dashed line in the bottom right WT panel shows the separation of the sheath cell–containing zone (right) from the vacuolated cell–containing zone (left). (B) Immunofluorescence analysis using the anti–type II collagen antibody along with fluorescence microscopic analysis of an area of the notochord of BBF2H7 -KO medaka carrying P brachyury - Venus at stage 27, where the vacuolization was completed. Bars, 10 µm. (C) Schematic representation of notochord structure after vacuolization. (D) Enlarged area surrounded by the yellow dashed line in A. p, perinotochordal basement membrane; s, sheath cell; v, vacuolated cell. Scale is the same as other insets.

    Techniques Used: Immunofluorescence, Staining, Fluorescence

    Abnormal vacuolization of notochord cells in BBF2H7 -KO medaka. (A) Fluorescence microscopic analysis of WT and BBF2H7 -KO medaka expressing P BBF2H7 - Venus at stages 27 and 31. Red arrowheads indicate bending sites. (B and C) BBF2H7 heterozygotes were crossed with ATF6α (B) or ATF6β heterozygotes (C) carrying P BiP - EGFP . The notochords of resulting fish with various genotypes were analyzed by a fluorescence microscope at stage 31 at the optimal exposure time (fluorescence intensity in ATF6α −/− and ATF6β −/− was ∼60 and 80%, respectively, of that in ATF6α +/− or ATF6β +/− medaka). Entire notochords were photographed, and the numbers of observed bendings were counted, as shown at the bottom. Data presented are means ± SD. (D) Immunofluorescence analyses of notochords at stage 27 of WT and BBF2H7 -KO medaka expressing P brachyury - Venus using an anti–type II collagen antibody. Insets are denoted by white dashed boxes. These are composite images. Bars: (A–C) 100 µm; (D) 30 µm.
    Figure Legend Snippet: Abnormal vacuolization of notochord cells in BBF2H7 -KO medaka. (A) Fluorescence microscopic analysis of WT and BBF2H7 -KO medaka expressing P BBF2H7 - Venus at stages 27 and 31. Red arrowheads indicate bending sites. (B and C) BBF2H7 heterozygotes were crossed with ATF6α (B) or ATF6β heterozygotes (C) carrying P BiP - EGFP . The notochords of resulting fish with various genotypes were analyzed by a fluorescence microscope at stage 31 at the optimal exposure time (fluorescence intensity in ATF6α −/− and ATF6β −/− was ∼60 and 80%, respectively, of that in ATF6α +/− or ATF6β +/− medaka). Entire notochords were photographed, and the numbers of observed bendings were counted, as shown at the bottom. Data presented are means ± SD. (D) Immunofluorescence analyses of notochords at stage 27 of WT and BBF2H7 -KO medaka expressing P brachyury - Venus using an anti–type II collagen antibody. Insets are denoted by white dashed boxes. These are composite images. Bars: (A–C) 100 µm; (D) 30 µm.

    Techniques Used: Fluorescence, Expressing, Fluorescence In Situ Hybridization, Microscopy, Immunofluorescence

    9) Product Images from "Measurements of heterotypic associations between cluster of differentiation CD74 and CD44 in human breast cancer-derived cells"

    Article Title: Measurements of heterotypic associations between cluster of differentiation CD74 and CD44 in human breast cancer-derived cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20922

    Confocal laser scanning microscopy images of intracellular staining of CD74 and CD44 in CAMA-1, MDA-MB-231 and MDA-MB-435 cells The cells were cultured in LabTek 8-well chambers at a density of 6 × 10 3 cells per well overnight. CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). 4′, 6-diamidino-2-phenylindole was used for nuclear staining (blue). Fluorochromes were acquired separately to evaluate the expression of CD74 and CD44 using the image-analysis software platform Fiji. Photomicrographs are representative of three independent experiments. Scale bar 10 μm.
    Figure Legend Snippet: Confocal laser scanning microscopy images of intracellular staining of CD74 and CD44 in CAMA-1, MDA-MB-231 and MDA-MB-435 cells The cells were cultured in LabTek 8-well chambers at a density of 6 × 10 3 cells per well overnight. CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). 4′, 6-diamidino-2-phenylindole was used for nuclear staining (blue). Fluorochromes were acquired separately to evaluate the expression of CD74 and CD44 using the image-analysis software platform Fiji. Photomicrographs are representative of three independent experiments. Scale bar 10 μm.

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Multiple Displacement Amplification, Cell Culture, Expressing, Software

    The expression of CD74 and CD44 receptors in immortalized normal breast luminal cells (226LDM) (A) Cell-surface and intracellular expression of CD74 and CD44 was acquired by flow cytometry using By2 (anti-CD74) and 156-3C11 (anti-CD44). Black-filled histograms represent the 226LDM cells stained with indicated antibody. Empty histograms show the isotype as negative controls. (B) Total protein of CD74 and CD44 was detected by Western blotting, and α-tubulin was used as a loading control. (C) Confocal images of CD74 and CD44 in 226LDM cells stained intracellularly: CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). Figures depict representative samples from duplicate experiments. Scale bar 10 μm.
    Figure Legend Snippet: The expression of CD74 and CD44 receptors in immortalized normal breast luminal cells (226LDM) (A) Cell-surface and intracellular expression of CD74 and CD44 was acquired by flow cytometry using By2 (anti-CD74) and 156-3C11 (anti-CD44). Black-filled histograms represent the 226LDM cells stained with indicated antibody. Empty histograms show the isotype as negative controls. (B) Total protein of CD74 and CD44 was detected by Western blotting, and α-tubulin was used as a loading control. (C) Confocal images of CD74 and CD44 in 226LDM cells stained intracellularly: CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). Figures depict representative samples from duplicate experiments. Scale bar 10 μm.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Western Blot

    10) Product Images from "Intravascular clearance of disseminating Cryptococcus neoformans in the brain can be improved by enhancing the recruitment of neutrophils"

    Article Title: Intravascular clearance of disseminating Cryptococcus neoformans in the brain can be improved by enhancing the recruitment of neutrophils

    Journal: European journal of immunology

    doi: 10.1002/eji.201546239

    Dramatic differences in vasculature structure between the lung and the brain. Mice were i.v. injected with 10 μg Alexa fluor 488 anti-PECAM-1 mAb for staining of the vasculature. The lung and the brain were imaged as described in the Materials
    Figure Legend Snippet: Dramatic differences in vasculature structure between the lung and the brain. Mice were i.v. injected with 10 μg Alexa fluor 488 anti-PECAM-1 mAb for staining of the vasculature. The lung and the brain were imaged as described in the Materials

    Techniques Used: Mouse Assay, Injection, Staining

    11) Product Images from "microRNAs (miR 9, 124, 155 and 224) transdifferentiate macrophages to neurons"

    Article Title: microRNAs (miR 9, 124, 155 and 224) transdifferentiate macrophages to neurons

    Journal: bioRxiv

    doi: 10.1101/2020.07.19.210633

    miR-mediated reprogramming involves stem cell-like intermediates. Macrophages were transfected with miRs twice and analysed for Stem cell markers, neuronal marker and pluripotency genes. (A) Flow cytometry analysis of transfected cells were done for NSE and Prominin (A’) NSE expressing cells are represented through histogram (n=3). (B) Relative density of the genes to β -actin were calculated and plotted as Heat Map considering Pu.1 in miR 224 as least and Oct4 in miR 155 as highest expression (C) miR 302 levels were measured RT-PCR in miR transfected cells. (D) Reprogrammed cells were selected on the basis of expression of CD11b and CD11b - cells were further selected for CD133. % of CD11b + ’ CD11b - CD133 + and CD11b - CD133 - cells were calculated to initial cells seeded and plotted. (E) CD133 + cells and CD11b - cells were further transfected once with miRs and stained for neuronal markers Nurr1 and Map2 and observed through immunofluorescence microscope. (400X magnification).
    Figure Legend Snippet: miR-mediated reprogramming involves stem cell-like intermediates. Macrophages were transfected with miRs twice and analysed for Stem cell markers, neuronal marker and pluripotency genes. (A) Flow cytometry analysis of transfected cells were done for NSE and Prominin (A’) NSE expressing cells are represented through histogram (n=3). (B) Relative density of the genes to β -actin were calculated and plotted as Heat Map considering Pu.1 in miR 224 as least and Oct4 in miR 155 as highest expression (C) miR 302 levels were measured RT-PCR in miR transfected cells. (D) Reprogrammed cells were selected on the basis of expression of CD11b and CD11b - cells were further selected for CD133. % of CD11b + ’ CD11b - CD133 + and CD11b - CD133 - cells were calculated to initial cells seeded and plotted. (E) CD133 + cells and CD11b - cells were further transfected once with miRs and stained for neuronal markers Nurr1 and Map2 and observed through immunofluorescence microscope. (400X magnification).

    Techniques Used: Transfection, Marker, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Immunofluorescence, Microscopy

    miR mimics transdifferentiate macrophages to induced Neuronal cells (iNCs). (A) Macrophages were transfected with miRs as indicated earlier for three times and gene expression was analysed by sq-RT PCR for Nestin and CD11b . (B) Immunofluorescence of a myeloid integrin, CD11c of reprogrammed cells. (B’) Cells expressing CD11c were counted in three random fields and plotted as % of CD11c + cells. Real time PCR analysis of Map2 (C), Tubb3 (D) and Mash1 (E) in neuron-like cells. (F and F’) Mature neural marker, NSE and NGFR were analysed by sq-RT PCR. (G and G’) Increase in neural markers NSE and Map2 was analysed using Western blot in iNCs. iNCs were fluorescently stained with various neuronal markers: (H) Map2 and Nurr1; (I) Nestin and NSE; (J) GFAP and (K) N-Cadherin. (400X magnification). (See supplementary figures S3A, S3B, S3C, S3D aslo) (Relative density was plotted using data from three individual experiments).
    Figure Legend Snippet: miR mimics transdifferentiate macrophages to induced Neuronal cells (iNCs). (A) Macrophages were transfected with miRs as indicated earlier for three times and gene expression was analysed by sq-RT PCR for Nestin and CD11b . (B) Immunofluorescence of a myeloid integrin, CD11c of reprogrammed cells. (B’) Cells expressing CD11c were counted in three random fields and plotted as % of CD11c + cells. Real time PCR analysis of Map2 (C), Tubb3 (D) and Mash1 (E) in neuron-like cells. (F and F’) Mature neural marker, NSE and NGFR were analysed by sq-RT PCR. (G and G’) Increase in neural markers NSE and Map2 was analysed using Western blot in iNCs. iNCs were fluorescently stained with various neuronal markers: (H) Map2 and Nurr1; (I) Nestin and NSE; (J) GFAP and (K) N-Cadherin. (400X magnification). (See supplementary figures S3A, S3B, S3C, S3D aslo) (Relative density was plotted using data from three individual experiments).

    Techniques Used: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Real-time Polymerase Chain Reaction, Marker, Western Blot, Staining

    Mouse primary macrophages were transdifferentiated to iNCs. iNCs generated from bone marrow derived were analysed for Map2, NSE (A) and Synapsin (B). iNCs generated from PBMCs express Map2 as visualized by immunofluorescence (C). (400X magnification)
    Figure Legend Snippet: Mouse primary macrophages were transdifferentiated to iNCs. iNCs generated from bone marrow derived were analysed for Map2, NSE (A) and Synapsin (B). iNCs generated from PBMCs express Map2 as visualized by immunofluorescence (C). (400X magnification)

    Techniques Used: Generated, Derivative Assay, Immunofluorescence

    miR transfections transdifferentiate macrophages to pure iNCs. (A) Flow cytometry analysis of 5 x 10 5 cells were done after every transfection for synapsin and Map2. (B) Efficacy of trans differentiation was plotted calculated from flow cytometry. (C) Trans differentiation efficacy and total no. of iNCs obtained after reprogramming were plotted.
    Figure Legend Snippet: miR transfections transdifferentiate macrophages to pure iNCs. (A) Flow cytometry analysis of 5 x 10 5 cells were done after every transfection for synapsin and Map2. (B) Efficacy of trans differentiation was plotted calculated from flow cytometry. (C) Trans differentiation efficacy and total no. of iNCs obtained after reprogramming were plotted.

    Techniques Used: Transfection, Flow Cytometry

    12) Product Images from "Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers"

    Article Title: Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

    Journal: eLife

    doi: 10.7554/eLife.25716

    Anti-DDR1 mAbs inhibit collagen-induced DDR1 clustering. Cos-7 cells expressing DDR1 were incubated with 10 μg/ml collagen I for 10 minutes at 37°C, left unstimulated, or incubated with collagen I in the presence of function-blocking anti-DDR1 mAbs. Cells were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488. Cells were imaged on a widefield microscope.
    Figure Legend Snippet: Anti-DDR1 mAbs inhibit collagen-induced DDR1 clustering. Cos-7 cells expressing DDR1 were incubated with 10 μg/ml collagen I for 10 minutes at 37°C, left unstimulated, or incubated with collagen I in the presence of function-blocking anti-DDR1 mAbs. Cells were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488. Cells were imaged on a widefield microscope.

    Techniques Used: Expressing, Incubation, Blocking Assay, Microscopy

    Function-blocking anti-DDR1 mAb inhibits collagen-induced DDR1 clustering. Wild-type DDR1b was transiently expressed in Cos-7 cells and either left unstimulated ( A ), stimulated with 10 μg/ml collagen I ( B ), or stimulated with 10 μg/ml collagen I in the presence of a function-blocking anti-DDR1 mAb at 10 μg/ml ( C ), for 10 min at 37°C. Cells from all conditions were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab, and mounting. Cells were imaged using a widefield microscope. White boxes in top rows indicate corresponding areas shown at higher magnification in lower rows. Scale bars, 30 μm (top rows) and 10 μm (bottom rows). DOI: http://dx.doi.org/10.7554/eLife.25716.019
    Figure Legend Snippet: Function-blocking anti-DDR1 mAb inhibits collagen-induced DDR1 clustering. Wild-type DDR1b was transiently expressed in Cos-7 cells and either left unstimulated ( A ), stimulated with 10 μg/ml collagen I ( B ), or stimulated with 10 μg/ml collagen I in the presence of a function-blocking anti-DDR1 mAb at 10 μg/ml ( C ), for 10 min at 37°C. Cells from all conditions were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab, and mounting. Cells were imaged using a widefield microscope. White boxes in top rows indicate corresponding areas shown at higher magnification in lower rows. Scale bars, 30 μm (top rows) and 10 μm (bottom rows). DOI: http://dx.doi.org/10.7554/eLife.25716.019

    Techniques Used: Blocking Assay, Incubation, Microscopy

    Cell surface expression of ectodomain-deletion constructs. ( A ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in Cos-7 cells. Cells were incubated on ice with mouse anti-Flag Ab, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab. Cells were imaged using a widefield microscope. Scale bars, 20 µm. ( B ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in HEK293 cells. Cells were stained on ice with rabbit anti-Flag Ab, followed by anti-rabbit Alexa-Fluor-488 conjugated secondary Ab and flow cytometry. Filled g ray histograms , secondary Ab only; open histograms , anti-Flag. DOI: http://dx.doi.org/10.7554/eLife.25716.010
    Figure Legend Snippet: Cell surface expression of ectodomain-deletion constructs. ( A ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in Cos-7 cells. Cells were incubated on ice with mouse anti-Flag Ab, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab. Cells were imaged using a widefield microscope. Scale bars, 20 µm. ( B ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in HEK293 cells. Cells were stained on ice with rabbit anti-Flag Ab, followed by anti-rabbit Alexa-Fluor-488 conjugated secondary Ab and flow cytometry. Filled g ray histograms , secondary Ab only; open histograms , anti-Flag. DOI: http://dx.doi.org/10.7554/eLife.25716.010

    Techniques Used: Expressing, Construct, Incubation, Microscopy, Staining, Flow Cytometry, Cytometry

    Collagen induces local activation of DDR1 and leads to recruitment of ectodomain-deletion DDR1 into the ligand-binding contact zone. ( A ) Wild-type DDR1b was transiently expressed in Cos-7 cells and stimulated with collagen-coated latex beads (3 μm diameter) for 2 or 4 hr at 37°C. Control beads were coated with BSA and incubated for 2 hr at 37°C. Cells were incubated on ice with a mouse monoclonal Ab against the D DR1 ectodomain, before fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs. DDR1 (green) and pY513 (red) staining are shown in the merge image (right panel). ( B ) The ectodomain deletion construct DDR1b-ΔECD (which contains an N-terminal Flag tag) and DDR1b-Y513F were expressed either alone or in combination in Cos-7 cells. Cells were stimulated with collagen-coated latex beads (3 μm diameter) for 2 hr at 37°C. Cells were incubated on ice with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IgG2b Abs, followed by fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488, anti-mouse IgG2b Alexa-Fluor-555, and anti-rabbit Alexa-Fluor-647 secondary Abs. Flag (green) and pY513 (red) staining are shown in the merge image (right panel). BF, Brightfield. Scale bars, 10 μm. DOI: http://dx.doi.org/10.7554/eLife.25716.020
    Figure Legend Snippet: Collagen induces local activation of DDR1 and leads to recruitment of ectodomain-deletion DDR1 into the ligand-binding contact zone. ( A ) Wild-type DDR1b was transiently expressed in Cos-7 cells and stimulated with collagen-coated latex beads (3 μm diameter) for 2 or 4 hr at 37°C. Control beads were coated with BSA and incubated for 2 hr at 37°C. Cells were incubated on ice with a mouse monoclonal Ab against the D DR1 ectodomain, before fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs. DDR1 (green) and pY513 (red) staining are shown in the merge image (right panel). ( B ) The ectodomain deletion construct DDR1b-ΔECD (which contains an N-terminal Flag tag) and DDR1b-Y513F were expressed either alone or in combination in Cos-7 cells. Cells were stimulated with collagen-coated latex beads (3 μm diameter) for 2 hr at 37°C. Cells were incubated on ice with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IgG2b Abs, followed by fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488, anti-mouse IgG2b Alexa-Fluor-555, and anti-rabbit Alexa-Fluor-647 secondary Abs. Flag (green) and pY513 (red) staining are shown in the merge image (right panel). BF, Brightfield. Scale bars, 10 μm. DOI: http://dx.doi.org/10.7554/eLife.25716.020

    Techniques Used: Activation Assay, Ligand Binding Assay, Incubation, Staining, Construct, FLAG-tag

    13) Product Images from "Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication"

    Article Title: Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication

    Journal: Virus Research

    doi: 10.1016/j.virusres.2020.197961

    Inhibition of ERK1/2 activation does not influence PDCoV-induced apoptosis. (A) ST cells were treated with DMSO or U0126 (100 μM) for 1 h prior to infection and then mock infected or infected with PDCoV in the presence of DMSO or U0126. The cells harvested at the indicated time points were subjected to dual Annexin V and PI labeling and analyzed by FACS. Lower left quadrants represent intact cells (Annexin V negative/PI negative); Lower right quadrants represent early apoptotic cells (Annexin V positive/PI negative); Upper right quadrants indicate late apoptotic or/and necrotic cells (Annexin V positive/PI positive); and Upper left quadrants indicate necrotic cells (Annexin V negative/PI positive). The figure is representative of three independent experiments. (B) The graph represents the percentage of each quadrant, and the non-significant percentages of Annexin V-negative and PI-positive cells were excluded. (C) The U0126-treated and infected cells were labeled with MitoTracker Red CMXRos (red), fixed at 12 hpi, and independently incubated with a primary antibody against Bax or cyt c (green). The cells were then counterstained with DAPI and examined under a confocal microscope at 800× magnification. PDCoV-specific CPEs were also photographed at 12 hpi under an inverted microscope at the magnification of 200× (left panels). Bax mitochondrial translocation is represented as the merger of Bax and mitochondrial marker (yellow), while the residual cytosolic localization is indicated by single staining signal (green). Conversely, cyt c cytosolic translocation is represented by single staining signal (green), and residual mitochondrial accumulation is indicated as the merger of cyt c and MitoTracker (yellow). The inset images are enlarged versions of parts of the picture.
    Figure Legend Snippet: Inhibition of ERK1/2 activation does not influence PDCoV-induced apoptosis. (A) ST cells were treated with DMSO or U0126 (100 μM) for 1 h prior to infection and then mock infected or infected with PDCoV in the presence of DMSO or U0126. The cells harvested at the indicated time points were subjected to dual Annexin V and PI labeling and analyzed by FACS. Lower left quadrants represent intact cells (Annexin V negative/PI negative); Lower right quadrants represent early apoptotic cells (Annexin V positive/PI negative); Upper right quadrants indicate late apoptotic or/and necrotic cells (Annexin V positive/PI positive); and Upper left quadrants indicate necrotic cells (Annexin V negative/PI positive). The figure is representative of three independent experiments. (B) The graph represents the percentage of each quadrant, and the non-significant percentages of Annexin V-negative and PI-positive cells were excluded. (C) The U0126-treated and infected cells were labeled with MitoTracker Red CMXRos (red), fixed at 12 hpi, and independently incubated with a primary antibody against Bax or cyt c (green). The cells were then counterstained with DAPI and examined under a confocal microscope at 800× magnification. PDCoV-specific CPEs were also photographed at 12 hpi under an inverted microscope at the magnification of 200× (left panels). Bax mitochondrial translocation is represented as the merger of Bax and mitochondrial marker (yellow), while the residual cytosolic localization is indicated by single staining signal (green). Conversely, cyt c cytosolic translocation is represented by single staining signal (green), and residual mitochondrial accumulation is indicated as the merger of cyt c and MitoTracker (yellow). The inset images are enlarged versions of parts of the picture.

    Techniques Used: Inhibition, Activation Assay, Infection, Labeling, FACS, Incubation, Microscopy, Inverted Microscopy, Translocation Assay, Marker, Staining

    14) Product Images from "Measurements of heterotypic associations between cluster of differentiation CD74 and CD44 in human breast cancer-derived cells"

    Article Title: Measurements of heterotypic associations between cluster of differentiation CD74 and CD44 in human breast cancer-derived cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20922

    Confocal laser scanning microscopy images of intracellular staining of CD74 and CD44 in CAMA-1, MDA-MB-231 and MDA-MB-435 cells The cells were cultured in LabTek 8-well chambers at a density of 6 × 10 3 cells per well overnight. CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). 4′, 6-diamidino-2-phenylindole was used for nuclear staining (blue). Fluorochromes were acquired separately to evaluate the expression of CD74 and CD44 using the image-analysis software platform Fiji. Photomicrographs are representative of three independent experiments. Scale bar 10 μm.
    Figure Legend Snippet: Confocal laser scanning microscopy images of intracellular staining of CD74 and CD44 in CAMA-1, MDA-MB-231 and MDA-MB-435 cells The cells were cultured in LabTek 8-well chambers at a density of 6 × 10 3 cells per well overnight. CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). 4′, 6-diamidino-2-phenylindole was used for nuclear staining (blue). Fluorochromes were acquired separately to evaluate the expression of CD74 and CD44 using the image-analysis software platform Fiji. Photomicrographs are representative of three independent experiments. Scale bar 10 μm.

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Multiple Displacement Amplification, Cell Culture, Expressing, Software

    The expression of CD74 and CD44 receptors in immortalized normal breast luminal cells (226LDM) (A) Cell-surface and intracellular expression of CD74 and CD44 was acquired by flow cytometry using By2 (anti-CD74) and 156-3C11 (anti-CD44). Black-filled histograms represent the 226LDM cells stained with indicated antibody. Empty histograms show the isotype as negative controls. (B) Total protein of CD74 and CD44 was detected by Western blotting, and α-tubulin was used as a loading control. (C) Confocal images of CD74 and CD44 in 226LDM cells stained intracellularly: CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). Figures depict representative samples from duplicate experiments. Scale bar 10 μm.
    Figure Legend Snippet: The expression of CD74 and CD44 receptors in immortalized normal breast luminal cells (226LDM) (A) Cell-surface and intracellular expression of CD74 and CD44 was acquired by flow cytometry using By2 (anti-CD74) and 156-3C11 (anti-CD44). Black-filled histograms represent the 226LDM cells stained with indicated antibody. Empty histograms show the isotype as negative controls. (B) Total protein of CD74 and CD44 was detected by Western blotting, and α-tubulin was used as a loading control. (C) Confocal images of CD74 and CD44 in 226LDM cells stained intracellularly: CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). Figures depict representative samples from duplicate experiments. Scale bar 10 μm.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Western Blot

    15) Product Images from "Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers"

    Article Title: Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

    Journal: eLife

    doi: 10.7554/eLife.25716

    Anti-DDR1 mAbs inhibit collagen-induced DDR1 clustering. Cos-7 cells expressing DDR1 were incubated with 10 μg/ml collagen I for 10 minutes at 37°C, left unstimulated, or incubated with collagen I in the presence of function-blocking anti-DDR1 mAbs. Cells were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488. Cells were imaged on a widefield microscope.
    Figure Legend Snippet: Anti-DDR1 mAbs inhibit collagen-induced DDR1 clustering. Cos-7 cells expressing DDR1 were incubated with 10 μg/ml collagen I for 10 minutes at 37°C, left unstimulated, or incubated with collagen I in the presence of function-blocking anti-DDR1 mAbs. Cells were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488. Cells were imaged on a widefield microscope.

    Techniques Used: Expressing, Incubation, Blocking Assay, Microscopy

    Function-blocking anti-DDR1 mAb inhibits collagen-induced DDR1 clustering. Wild-type DDR1b was transiently expressed in Cos-7 cells and either left unstimulated ( A ), stimulated with 10 μg/ml collagen I ( B ), or stimulated with 10 μg/ml collagen I in the presence of a function-blocking anti-DDR1 mAb at 10 μg/ml ( C ), for 10 min at 37°C. Cells from all conditions were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab, and mounting. Cells were imaged using a widefield microscope. White boxes in top rows indicate corresponding areas shown at higher magnification in lower rows. Scale bars, 30 μm (top rows) and 10 μm (bottom rows). DOI: http://dx.doi.org/10.7554/eLife.25716.019
    Figure Legend Snippet: Function-blocking anti-DDR1 mAb inhibits collagen-induced DDR1 clustering. Wild-type DDR1b was transiently expressed in Cos-7 cells and either left unstimulated ( A ), stimulated with 10 μg/ml collagen I ( B ), or stimulated with 10 μg/ml collagen I in the presence of a function-blocking anti-DDR1 mAb at 10 μg/ml ( C ), for 10 min at 37°C. Cells from all conditions were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab, and mounting. Cells were imaged using a widefield microscope. White boxes in top rows indicate corresponding areas shown at higher magnification in lower rows. Scale bars, 30 μm (top rows) and 10 μm (bottom rows). DOI: http://dx.doi.org/10.7554/eLife.25716.019

    Techniques Used: Blocking Assay, Incubation, Microscopy

    Cell surface expression of ectodomain-deletion constructs. ( A ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in Cos-7 cells. Cells were incubated on ice with mouse anti-Flag Ab, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab. Cells were imaged using a widefield microscope. Scale bars, 20 µm. ( B ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in HEK293 cells. Cells were stained on ice with rabbit anti-Flag Ab, followed by anti-rabbit Alexa-Fluor-488 conjugated secondary Ab and flow cytometry. Filled g ray histograms , secondary Ab only; open histograms , anti-Flag. DOI: http://dx.doi.org/10.7554/eLife.25716.010
    Figure Legend Snippet: Cell surface expression of ectodomain-deletion constructs. ( A ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in Cos-7 cells. Cells were incubated on ice with mouse anti-Flag Ab, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab. Cells were imaged using a widefield microscope. Scale bars, 20 µm. ( B ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in HEK293 cells. Cells were stained on ice with rabbit anti-Flag Ab, followed by anti-rabbit Alexa-Fluor-488 conjugated secondary Ab and flow cytometry. Filled g ray histograms , secondary Ab only; open histograms , anti-Flag. DOI: http://dx.doi.org/10.7554/eLife.25716.010

    Techniques Used: Expressing, Construct, Incubation, Microscopy, Staining, Flow Cytometry, Cytometry

    Collagen induces local activation of DDR1 and leads to recruitment of ectodomain-deletion DDR1 into the ligand-binding contact zone. ( A ) Wild-type DDR1b was transiently expressed in Cos-7 cells and stimulated with collagen-coated latex beads (3 μm diameter) for 2 or 4 hr at 37°C. Control beads were coated with BSA and incubated for 2 hr at 37°C. Cells were incubated on ice with a mouse monoclonal Ab against the D DR1 ectodomain, before fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs. DDR1 (green) and pY513 (red) staining are shown in the merge image (right panel). ( B ) The ectodomain deletion construct DDR1b-ΔECD (which contains an N-terminal Flag tag) and DDR1b-Y513F were expressed either alone or in combination in Cos-7 cells. Cells were stimulated with collagen-coated latex beads (3 μm diameter) for 2 hr at 37°C. Cells were incubated on ice with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IgG2b Abs, followed by fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488, anti-mouse IgG2b Alexa-Fluor-555, and anti-rabbit Alexa-Fluor-647 secondary Abs. Flag (green) and pY513 (red) staining are shown in the merge image (right panel). BF, Brightfield. Scale bars, 10 μm. DOI: http://dx.doi.org/10.7554/eLife.25716.020
    Figure Legend Snippet: Collagen induces local activation of DDR1 and leads to recruitment of ectodomain-deletion DDR1 into the ligand-binding contact zone. ( A ) Wild-type DDR1b was transiently expressed in Cos-7 cells and stimulated with collagen-coated latex beads (3 μm diameter) for 2 or 4 hr at 37°C. Control beads were coated with BSA and incubated for 2 hr at 37°C. Cells were incubated on ice with a mouse monoclonal Ab against the D DR1 ectodomain, before fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs. DDR1 (green) and pY513 (red) staining are shown in the merge image (right panel). ( B ) The ectodomain deletion construct DDR1b-ΔECD (which contains an N-terminal Flag tag) and DDR1b-Y513F were expressed either alone or in combination in Cos-7 cells. Cells were stimulated with collagen-coated latex beads (3 μm diameter) for 2 hr at 37°C. Cells were incubated on ice with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IgG2b Abs, followed by fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488, anti-mouse IgG2b Alexa-Fluor-555, and anti-rabbit Alexa-Fluor-647 secondary Abs. Flag (green) and pY513 (red) staining are shown in the merge image (right panel). BF, Brightfield. Scale bars, 10 μm. DOI: http://dx.doi.org/10.7554/eLife.25716.020

    Techniques Used: Activation Assay, Ligand Binding Assay, Incubation, Staining, Construct, FLAG-tag

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    Thermo Fisher alexa flour 488 conjugated anti mouse igg h l
    Establishment of monkey podoplanin-neutralizing antibodies (A) Purification of a monkey podoplanin (mkyPDPN) immunogen to establish a hybridoma secreting anti-PDPN monoclonal antibodies (mAbs). A mkyPDPN cDNA region encoding amino acids 76–89 (226–267 bp) was tandemly connected 21 times. The cDNA fragment was inserted into a pGEX-6P-3 vector, and a glutathione S-transferase (GST)-tagged mkyPDPN peptide (76–89 aa) produced by BL21 (DE3) E. coli was purified using glutathione sepharose. BALB/c mice were injected with the GST-tagged peptide, after which their splenocytes were fused with mouse myeloma P3U1 cells using polyethylene glycol. Hybridoma screening and antibody purification from ascites were performed. (B) CHO cells transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT) were treated with PBS (closed areas) or antibodies (open areas), including anti-PDPN mAb D2-40, 1F6, 2F7, or 3F4 to measure PDPN expression levels. (C) GST-tagged recombinant mkyPDPN protein (WT) and its point mutants were expressed in E. coli . Cell lysates were electrophoresed and immunoblotted with antibodies to PDPN (1F6, 2F7, 3F4) or GST. The PLAG4 domain is indicated by red letters. (D) CHO/mock, CHO/mkyPDPN-WT, or CHO/hPDPN-WT cells were incubated with 100 μg/mL of control IgG1 or anti-PDPN antibodies 1F6, 2F7, or 3F4, followed by incubation with 1 μg/mL of hCLEC-2-(His) 10 (open areas: control IgG-treated samples; green areas: anti-PDPN mAb-treated samples). After washing, cells were further incubated with <t>Alexa</t> Flour 488-conjugated anti-penta-His second antibody. CLEC-2 binding was measured by flow cytometry. Gray areas indicate the fluorescence intensity of samples not treated with CLEC-2. (E) CHO/mkyPDPN-WT cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs followed by incubation with mouse platelet-rich plasma (PRP). The aggregation rate was estimated using an aggregometer. (F) PLAG3 domain-deleted mkyPDPN mutant cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs, followed by incubation with mouse PRP. The aggregation rate was estimated using an aggregometer.
    Alexa Flour 488 Conjugated Anti Mouse Igg H L, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Establishment of monkey podoplanin-neutralizing antibodies (A) Purification of a monkey podoplanin (mkyPDPN) immunogen to establish a hybridoma secreting anti-PDPN monoclonal antibodies (mAbs). A mkyPDPN cDNA region encoding amino acids 76–89 (226–267 bp) was tandemly connected 21 times. The cDNA fragment was inserted into a pGEX-6P-3 vector, and a glutathione S-transferase (GST)-tagged mkyPDPN peptide (76–89 aa) produced by BL21 (DE3) E. coli was purified using glutathione sepharose. BALB/c mice were injected with the GST-tagged peptide, after which their splenocytes were fused with mouse myeloma P3U1 cells using polyethylene glycol. Hybridoma screening and antibody purification from ascites were performed. (B) CHO cells transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT) were treated with PBS (closed areas) or antibodies (open areas), including anti-PDPN mAb D2-40, 1F6, 2F7, or 3F4 to measure PDPN expression levels. (C) GST-tagged recombinant mkyPDPN protein (WT) and its point mutants were expressed in E. coli . Cell lysates were electrophoresed and immunoblotted with antibodies to PDPN (1F6, 2F7, 3F4) or GST. The PLAG4 domain is indicated by red letters. (D) CHO/mock, CHO/mkyPDPN-WT, or CHO/hPDPN-WT cells were incubated with 100 μg/mL of control IgG1 or anti-PDPN antibodies 1F6, 2F7, or 3F4, followed by incubation with 1 μg/mL of hCLEC-2-(His) 10 (open areas: control IgG-treated samples; green areas: anti-PDPN mAb-treated samples). After washing, cells were further incubated with Alexa Flour 488-conjugated anti-penta-His second antibody. CLEC-2 binding was measured by flow cytometry. Gray areas indicate the fluorescence intensity of samples not treated with CLEC-2. (E) CHO/mkyPDPN-WT cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs followed by incubation with mouse platelet-rich plasma (PRP). The aggregation rate was estimated using an aggregometer. (F) PLAG3 domain-deleted mkyPDPN mutant cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs, followed by incubation with mouse PRP. The aggregation rate was estimated using an aggregometer.

    Journal: Oncotarget

    Article Title: A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey (Macaca fascicularis)

    doi: 10.18632/oncotarget.26055

    Figure Lengend Snippet: Establishment of monkey podoplanin-neutralizing antibodies (A) Purification of a monkey podoplanin (mkyPDPN) immunogen to establish a hybridoma secreting anti-PDPN monoclonal antibodies (mAbs). A mkyPDPN cDNA region encoding amino acids 76–89 (226–267 bp) was tandemly connected 21 times. The cDNA fragment was inserted into a pGEX-6P-3 vector, and a glutathione S-transferase (GST)-tagged mkyPDPN peptide (76–89 aa) produced by BL21 (DE3) E. coli was purified using glutathione sepharose. BALB/c mice were injected with the GST-tagged peptide, after which their splenocytes were fused with mouse myeloma P3U1 cells using polyethylene glycol. Hybridoma screening and antibody purification from ascites were performed. (B) CHO cells transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT) were treated with PBS (closed areas) or antibodies (open areas), including anti-PDPN mAb D2-40, 1F6, 2F7, or 3F4 to measure PDPN expression levels. (C) GST-tagged recombinant mkyPDPN protein (WT) and its point mutants were expressed in E. coli . Cell lysates were electrophoresed and immunoblotted with antibodies to PDPN (1F6, 2F7, 3F4) or GST. The PLAG4 domain is indicated by red letters. (D) CHO/mock, CHO/mkyPDPN-WT, or CHO/hPDPN-WT cells were incubated with 100 μg/mL of control IgG1 or anti-PDPN antibodies 1F6, 2F7, or 3F4, followed by incubation with 1 μg/mL of hCLEC-2-(His) 10 (open areas: control IgG-treated samples; green areas: anti-PDPN mAb-treated samples). After washing, cells were further incubated with Alexa Flour 488-conjugated anti-penta-His second antibody. CLEC-2 binding was measured by flow cytometry. Gray areas indicate the fluorescence intensity of samples not treated with CLEC-2. (E) CHO/mkyPDPN-WT cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs followed by incubation with mouse platelet-rich plasma (PRP). The aggregation rate was estimated using an aggregometer. (F) PLAG3 domain-deleted mkyPDPN mutant cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs, followed by incubation with mouse PRP. The aggregation rate was estimated using an aggregometer.

    Article Snippet: Flow cytometric analysis To analyze podoplanin expression, cells were harvested and treated with 1 μg/mL of anti-podoplanin antibodies (Sigma-Aldrich), followed by incubation with Alexa Flour 488-conjugated anti-mouse IgG (H+L) (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Purification, Plasmid Preparation, Produced, Mouse Assay, Injection, Antibody Purification, Transfection, Expressing, Recombinant, Incubation, Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Mutagenesis

    PLAG4 domain contributes to monkey podoplanin-induced platelet aggregation (A) CHO cells stably transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT), or PLAG domain-deleted podoplanin mutants (mkyPDPN−ΔPLAG3, −ΔPLAG4, and −ΔPLAG3+4) were treated with PBS (closed areas) or anti-podoplanin mAb (D2-40; open areas) to measure PDPN expression levels. The ability to bind with human or mouse C-type lectin-like receptor 2 (CLEC-2) was compared with PBS (closed areas), hCLEC-2-(His) 10 (0.4 μg/ml), or mCLEC-2-(His) 10 (5 μg/ml) (open areas). After washing, cells were incubated with Alexa Flour 488-conjugated second antibody. Flow cytometry data (upper) and quantitative graphs (lower) are shown. Each value in the lower graphs is the mean ± SD (N = 3) of peak values normalized to that of CHO/PDPN-WT. * P

    Journal: Oncotarget

    Article Title: A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey (Macaca fascicularis)

    doi: 10.18632/oncotarget.26055

    Figure Lengend Snippet: PLAG4 domain contributes to monkey podoplanin-induced platelet aggregation (A) CHO cells stably transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT), or PLAG domain-deleted podoplanin mutants (mkyPDPN−ΔPLAG3, −ΔPLAG4, and −ΔPLAG3+4) were treated with PBS (closed areas) or anti-podoplanin mAb (D2-40; open areas) to measure PDPN expression levels. The ability to bind with human or mouse C-type lectin-like receptor 2 (CLEC-2) was compared with PBS (closed areas), hCLEC-2-(His) 10 (0.4 μg/ml), or mCLEC-2-(His) 10 (5 μg/ml) (open areas). After washing, cells were incubated with Alexa Flour 488-conjugated second antibody. Flow cytometry data (upper) and quantitative graphs (lower) are shown. Each value in the lower graphs is the mean ± SD (N = 3) of peak values normalized to that of CHO/PDPN-WT. * P

    Article Snippet: Flow cytometric analysis To analyze podoplanin expression, cells were harvested and treated with 1 μg/mL of anti-podoplanin antibodies (Sigma-Aldrich), followed by incubation with Alexa Flour 488-conjugated anti-mouse IgG (H+L) (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Incubation, Flow Cytometry, Cytometry

    Establishment of monkey podoplanin-neutralizing antibodies (A) Purification of a monkey podoplanin (mkyPDPN) immunogen to establish a hybridoma secreting anti-PDPN monoclonal antibodies (mAbs). A mkyPDPN cDNA region encoding amino acids 76–89 (226–267 bp) was tandemly connected 21 times. The cDNA fragment was inserted into a pGEX-6P-3 vector, and a glutathione S-transferase (GST)-tagged mkyPDPN peptide (76–89 aa) produced by BL21 (DE3) E. coli was purified using glutathione sepharose. BALB/c mice were injected with the GST-tagged peptide, after which their splenocytes were fused with mouse myeloma P3U1 cells using polyethylene glycol. Hybridoma screening and antibody purification from ascites were performed. (B) CHO cells transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT) were treated with PBS (closed areas) or antibodies (open areas), including anti-PDPN mAb D2-40, 1F6, 2F7, or 3F4 to measure PDPN expression levels. (C) GST-tagged recombinant mkyPDPN protein (WT) and its point mutants were expressed in E. coli . Cell lysates were electrophoresed and immunoblotted with antibodies to PDPN (1F6, 2F7, 3F4) or GST. The PLAG4 domain is indicated by red letters. (D) CHO/mock, CHO/mkyPDPN-WT, or CHO/hPDPN-WT cells were incubated with 100 μg/mL of control IgG1 or anti-PDPN antibodies 1F6, 2F7, or 3F4, followed by incubation with 1 μg/mL of hCLEC-2-(His) 10 (open areas: control IgG-treated samples; green areas: anti-PDPN mAb-treated samples). After washing, cells were further incubated with Alexa Flour 488-conjugated anti-penta-His second antibody. CLEC-2 binding was measured by flow cytometry. Gray areas indicate the fluorescence intensity of samples not treated with CLEC-2. (E) CHO/mkyPDPN-WT cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs followed by incubation with mouse platelet-rich plasma (PRP). The aggregation rate was estimated using an aggregometer. (F) PLAG3 domain-deleted mkyPDPN mutant cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs, followed by incubation with mouse PRP. The aggregation rate was estimated using an aggregometer.

    Journal: Oncotarget

    Article Title: A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey (Macaca fascicularis)

    doi: 10.18632/oncotarget.26055

    Figure Lengend Snippet: Establishment of monkey podoplanin-neutralizing antibodies (A) Purification of a monkey podoplanin (mkyPDPN) immunogen to establish a hybridoma secreting anti-PDPN monoclonal antibodies (mAbs). A mkyPDPN cDNA region encoding amino acids 76–89 (226–267 bp) was tandemly connected 21 times. The cDNA fragment was inserted into a pGEX-6P-3 vector, and a glutathione S-transferase (GST)-tagged mkyPDPN peptide (76–89 aa) produced by BL21 (DE3) E. coli was purified using glutathione sepharose. BALB/c mice were injected with the GST-tagged peptide, after which their splenocytes were fused with mouse myeloma P3U1 cells using polyethylene glycol. Hybridoma screening and antibody purification from ascites were performed. (B) CHO cells transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT) were treated with PBS (closed areas) or antibodies (open areas), including anti-PDPN mAb D2-40, 1F6, 2F7, or 3F4 to measure PDPN expression levels. (C) GST-tagged recombinant mkyPDPN protein (WT) and its point mutants were expressed in E. coli . Cell lysates were electrophoresed and immunoblotted with antibodies to PDPN (1F6, 2F7, 3F4) or GST. The PLAG4 domain is indicated by red letters. (D) CHO/mock, CHO/mkyPDPN-WT, or CHO/hPDPN-WT cells were incubated with 100 μg/mL of control IgG1 or anti-PDPN antibodies 1F6, 2F7, or 3F4, followed by incubation with 1 μg/mL of hCLEC-2-(His) 10 (open areas: control IgG-treated samples; green areas: anti-PDPN mAb-treated samples). After washing, cells were further incubated with Alexa Flour 488-conjugated anti-penta-His second antibody. CLEC-2 binding was measured by flow cytometry. Gray areas indicate the fluorescence intensity of samples not treated with CLEC-2. (E) CHO/mkyPDPN-WT cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs followed by incubation with mouse platelet-rich plasma (PRP). The aggregation rate was estimated using an aggregometer. (F) PLAG3 domain-deleted mkyPDPN mutant cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs, followed by incubation with mouse PRP. The aggregation rate was estimated using an aggregometer.

    Article Snippet: To analyze podoplanin expression, cells were harvested and treated with 1 μg/mL of anti-podoplanin antibodies (Sigma-Aldrich), followed by incubation with Alexa Flour 488-conjugated anti-mouse IgG (H+L) (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Purification, Plasmid Preparation, Produced, Mouse Assay, Injection, Antibody Purification, Transfection, Expressing, Recombinant, Incubation, Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Mutagenesis

    PLAG4 domain contributes to monkey podoplanin-induced platelet aggregation (A) CHO cells stably transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT), or PLAG domain-deleted podoplanin mutants (mkyPDPN−ΔPLAG3, −ΔPLAG4, and −ΔPLAG3+4) were treated with PBS (closed areas) or anti-podoplanin mAb (D2-40; open areas) to measure PDPN expression levels. The ability to bind with human or mouse C-type lectin-like receptor 2 (CLEC-2) was compared with PBS (closed areas), hCLEC-2-(His) 10 (0.4 μg/ml), or mCLEC-2-(His) 10 (5 μg/ml) (open areas). After washing, cells were incubated with Alexa Flour 488-conjugated second antibody. Flow cytometry data (upper) and quantitative graphs (lower) are shown. Each value in the lower graphs is the mean ± SD (N = 3) of peak values normalized to that of CHO/PDPN-WT. * P

    Journal: Oncotarget

    Article Title: A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey (Macaca fascicularis)

    doi: 10.18632/oncotarget.26055

    Figure Lengend Snippet: PLAG4 domain contributes to monkey podoplanin-induced platelet aggregation (A) CHO cells stably transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT), or PLAG domain-deleted podoplanin mutants (mkyPDPN−ΔPLAG3, −ΔPLAG4, and −ΔPLAG3+4) were treated with PBS (closed areas) or anti-podoplanin mAb (D2-40; open areas) to measure PDPN expression levels. The ability to bind with human or mouse C-type lectin-like receptor 2 (CLEC-2) was compared with PBS (closed areas), hCLEC-2-(His) 10 (0.4 μg/ml), or mCLEC-2-(His) 10 (5 μg/ml) (open areas). After washing, cells were incubated with Alexa Flour 488-conjugated second antibody. Flow cytometry data (upper) and quantitative graphs (lower) are shown. Each value in the lower graphs is the mean ± SD (N = 3) of peak values normalized to that of CHO/PDPN-WT. * P

    Article Snippet: To analyze podoplanin expression, cells were harvested and treated with 1 μg/mL of anti-podoplanin antibodies (Sigma-Aldrich), followed by incubation with Alexa Flour 488-conjugated anti-mouse IgG (H+L) (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Incubation, Flow Cytometry, Cytometry