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Jackson Immuno donkey f
Donkey F, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 9 article reviews
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donkey f - by Bioz Stars, 2020-09
92/100 stars

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Related Articles

Enzyme-linked Immunosorbent Assay:

Article Title: Immunization-elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability
Article Snippet: .. Maxisorp ELISA plates (Nunc) were coated with 2 μg/mL of GP proteins (EBOV, SUDV or BDBV) for detecting GP binding or AffiniPure Donkey anti-Human IgG, Fcγ fragment (Jackson ImmunoResearch Labs) for normalizing IgG expression, respectively in PBS (pH 7.4) at 4°C overnight. .. After incubation with blocking buffer (BB, 2% non-fat milk, 5% FBS in PBS), the WT or mutant supernatant in five-fold serial dilution in BB was added and incubated for 1 hour at 37°C.

Concentration Assay:

Article Title: Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG
Article Snippet: .. A 384-well microtiter plate was coated with 25 µL of donkey F(ab')2 anti-huIgG (Fc fragment specific) (Jackson ImmunoResearch) at a concentration of 0.25 µg/mL in PBS. .. The plate was incubated at 2–8°C overnight and then washed three times with wash buffer.

Binding Assay:

Article Title: Immunization-elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability
Article Snippet: .. Maxisorp ELISA plates (Nunc) were coated with 2 μg/mL of GP proteins (EBOV, SUDV or BDBV) for detecting GP binding or AffiniPure Donkey anti-Human IgG, Fcγ fragment (Jackson ImmunoResearch Labs) for normalizing IgG expression, respectively in PBS (pH 7.4) at 4°C overnight. .. After incubation with blocking buffer (BB, 2% non-fat milk, 5% FBS in PBS), the WT or mutant supernatant in five-fold serial dilution in BB was added and incubated for 1 hour at 37°C.

Expressing:

Article Title: Immunization-elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability
Article Snippet: .. Maxisorp ELISA plates (Nunc) were coated with 2 μg/mL of GP proteins (EBOV, SUDV or BDBV) for detecting GP binding or AffiniPure Donkey anti-Human IgG, Fcγ fragment (Jackson ImmunoResearch Labs) for normalizing IgG expression, respectively in PBS (pH 7.4) at 4°C overnight. .. After incubation with blocking buffer (BB, 2% non-fat milk, 5% FBS in PBS), the WT or mutant supernatant in five-fold serial dilution in BB was added and incubated for 1 hour at 37°C.

Staining:

Article Title: Identifying the cellular location of brain cytoplasmic 200 RNA using an RNA-recognizing antibody
Article Snippet: .. The MabBC200-A3 antibody (500 ng/100 ul) was used to stain BC200 RNA, following which it was visualized using Cy™2 AffiniPure Donkey Anti-Human IgG (1:200, Jackson Immunoresearch). .. Imaging was performed using a confocal Zeiss microscope (Zeiss LSM780).

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  • 94
    Jackson Immuno rhodamine anti gp igg
    NF-Y activity is critical for endogenous Bim expression in sympathetic neurons. ( a ) The NF-YA dominant negative mutant, YA13 m29, localises to the nucleus in <t>microinjected</t> sympathetic neurons. Cells were injected with the YA13 m29 expression vector at 20 ng/ μ l along with 2.5 μ g/ μ l of gp <t>IgG.</t> After 24 h, neurons were fixed and then stained with Hoechst dye, to visualise the nuclei, and antibodies to gp IgG (to identify injected cells) and NF-YA (H-209) to detect YA13 m29. Representative images of several experiments are shown. The scale bar represents 20 μ m. ( b ) Sympathetic neurons were co-microinjected with bim -LUC at 10 ng/ μ l along with pRL-TK (5 ng/ μ l), plus YA13 m29 or the control empty vector pSG5 at 20 ng/ μ l. The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (−NGF) for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =4. Bim -LUC is activated significantly following NGF withdrawal when co-injected with pSG5 ( P =0.015). When bim -LUC is co-injected with YA13 m29, there is a significant decrease in basal promoter level ( P =0.003) and the induction of bim -LUC, following NGF withdrawal, is abolished. ( c ) YA13 m29 reduces endogenous Bim expression in microinjected sympathetic neurons. Cells were injected with the YA13 m29 construct or the control empty vector pSG5 at 20 ng/ μ l along with 2.5 μ g/ μ l of gp IgG. The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (−NGF) for 16 h. Following NGF withdrawal, neurons were fixed and then stained with Hoechst dye and antibodies to gp IgG and Bim (#2819). In the presence of NGF, cells either expressed no Bim protein or low levels of Bim protein, whereas following NGF withdrawal many cells expressed high levels of Bim protein. Therefore, cells were scored as no/low Bim expression (comparable to +NGF conditions) or high Bim expression. The white arrow indicates a sympathetic neuron injected with pSG5 that is expressing high levels of Bim protein. The orange arrow indicates a sympathetic neuron injected with YA13 m29 that is expressing low levels of Bim protein. Representative images are shown. The bar represents 20 μ m. ( d ) Quantitation of immunocytochemistry in ( c ). Data are presented as the mean±S.E., n =6. White bars indicate no or low Bim staining and black bars indicate high Bim staining. Microinjection of the YA13 m29 construct significantly decreases Bim expression following NGF withdrawal compared with the control pSG5 ( P =0.002). ( *** P
    Rhodamine Anti Gp Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhodamine anti gp igg/product/Jackson Immuno
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rhodamine anti gp igg - by Bioz Stars, 2020-09
    94/100 stars
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    99
    Jackson Immuno fitc conjugated secondary antibodies
    Caspase-4 interacts by cathepsin G by Td92 treatment. ( a ) HGFs were treated with Td92 (10 μ g/ml) or Td-G (5 μ g/ml) for 1 h. Stimulated cells were incubated with anti-cathepsin G antibody and anti-caspase-4 antibody followed by incubation with <t>Cy3-</t> and <t>FITC-conjugated</t> secondary antibodies, respectively. Cells were imaged by confocal laser scanning microscopy. Scale bars represent 50 μ m. ( b ) HGFs were pretreated with cathepsin G inhibitor I (CatG I) for 30 min or transfected with control siRNA or siRNA specific for cathepsin G for 24 h prior to stimulation with Td92 for 1 h. Images of 10 microscopic fields per well were taken at × 200 magnification, and the percentage of coaggregates was calculated relative to total cells using NIH ImageJ software. At least 500 cells were counted for each experimental condition. Data are shown as the means±S.D. of three independent experiments. * P
    Fitc Conjugated Secondary Antibodies, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fitc conjugated secondary antibodies/product/Jackson Immuno
    Average 99 stars, based on 234 article reviews
    Price from $9.99 to $1999.99
    fitc conjugated secondary antibodies - by Bioz Stars, 2020-09
    99/100 stars
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    Image Search Results


    NF-Y activity is critical for endogenous Bim expression in sympathetic neurons. ( a ) The NF-YA dominant negative mutant, YA13 m29, localises to the nucleus in microinjected sympathetic neurons. Cells were injected with the YA13 m29 expression vector at 20 ng/ μ l along with 2.5 μ g/ μ l of gp IgG. After 24 h, neurons were fixed and then stained with Hoechst dye, to visualise the nuclei, and antibodies to gp IgG (to identify injected cells) and NF-YA (H-209) to detect YA13 m29. Representative images of several experiments are shown. The scale bar represents 20 μ m. ( b ) Sympathetic neurons were co-microinjected with bim -LUC at 10 ng/ μ l along with pRL-TK (5 ng/ μ l), plus YA13 m29 or the control empty vector pSG5 at 20 ng/ μ l. The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (−NGF) for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =4. Bim -LUC is activated significantly following NGF withdrawal when co-injected with pSG5 ( P =0.015). When bim -LUC is co-injected with YA13 m29, there is a significant decrease in basal promoter level ( P =0.003) and the induction of bim -LUC, following NGF withdrawal, is abolished. ( c ) YA13 m29 reduces endogenous Bim expression in microinjected sympathetic neurons. Cells were injected with the YA13 m29 construct or the control empty vector pSG5 at 20 ng/ μ l along with 2.5 μ g/ μ l of gp IgG. The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (−NGF) for 16 h. Following NGF withdrawal, neurons were fixed and then stained with Hoechst dye and antibodies to gp IgG and Bim (#2819). In the presence of NGF, cells either expressed no Bim protein or low levels of Bim protein, whereas following NGF withdrawal many cells expressed high levels of Bim protein. Therefore, cells were scored as no/low Bim expression (comparable to +NGF conditions) or high Bim expression. The white arrow indicates a sympathetic neuron injected with pSG5 that is expressing high levels of Bim protein. The orange arrow indicates a sympathetic neuron injected with YA13 m29 that is expressing low levels of Bim protein. Representative images are shown. The bar represents 20 μ m. ( d ) Quantitation of immunocytochemistry in ( c ). Data are presented as the mean±S.E., n =6. White bars indicate no or low Bim staining and black bars indicate high Bim staining. Microinjection of the YA13 m29 construct significantly decreases Bim expression following NGF withdrawal compared with the control pSG5 ( P =0.002). ( *** P

    Journal: Cell Death and Differentiation

    Article Title: NF-Y is essential for expression of the proapoptotic bim gene in sympathetic neurons

    doi: 10.1038/cdd.2010.166

    Figure Lengend Snippet: NF-Y activity is critical for endogenous Bim expression in sympathetic neurons. ( a ) The NF-YA dominant negative mutant, YA13 m29, localises to the nucleus in microinjected sympathetic neurons. Cells were injected with the YA13 m29 expression vector at 20 ng/ μ l along with 2.5 μ g/ μ l of gp IgG. After 24 h, neurons were fixed and then stained with Hoechst dye, to visualise the nuclei, and antibodies to gp IgG (to identify injected cells) and NF-YA (H-209) to detect YA13 m29. Representative images of several experiments are shown. The scale bar represents 20 μ m. ( b ) Sympathetic neurons were co-microinjected with bim -LUC at 10 ng/ μ l along with pRL-TK (5 ng/ μ l), plus YA13 m29 or the control empty vector pSG5 at 20 ng/ μ l. The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (−NGF) for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =4. Bim -LUC is activated significantly following NGF withdrawal when co-injected with pSG5 ( P =0.015). When bim -LUC is co-injected with YA13 m29, there is a significant decrease in basal promoter level ( P =0.003) and the induction of bim -LUC, following NGF withdrawal, is abolished. ( c ) YA13 m29 reduces endogenous Bim expression in microinjected sympathetic neurons. Cells were injected with the YA13 m29 construct or the control empty vector pSG5 at 20 ng/ μ l along with 2.5 μ g/ μ l of gp IgG. The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (−NGF) for 16 h. Following NGF withdrawal, neurons were fixed and then stained with Hoechst dye and antibodies to gp IgG and Bim (#2819). In the presence of NGF, cells either expressed no Bim protein or low levels of Bim protein, whereas following NGF withdrawal many cells expressed high levels of Bim protein. Therefore, cells were scored as no/low Bim expression (comparable to +NGF conditions) or high Bim expression. The white arrow indicates a sympathetic neuron injected with pSG5 that is expressing high levels of Bim protein. The orange arrow indicates a sympathetic neuron injected with YA13 m29 that is expressing low levels of Bim protein. Representative images are shown. The bar represents 20 μ m. ( d ) Quantitation of immunocytochemistry in ( c ). Data are presented as the mean±S.E., n =6. White bars indicate no or low Bim staining and black bars indicate high Bim staining. Microinjection of the YA13 m29 construct significantly decreases Bim expression following NGF withdrawal compared with the control pSG5 ( P =0.002). ( *** P

    Article Snippet: Secondary antibodies were a FITC-conjugated anti-rabbit IgG, and a rhodamine anti-gp IgG if neurons were microinjected (both from Jackson ImmunoResearch).

    Techniques: Activity Assay, Expressing, Dominant Negative Mutation, Injection, Plasmid Preparation, Staining, Luciferase, Construct, Quantitation Assay, Immunocytochemistry

    CBP and p300 are important for activation of the bim promoter in sympathetic neurons. ( a ) Co-microinjection of antibodies specific to p300 or CBP with bim -LUC. Sympathetic neurons were microinjected with bim -LUC at 10 ng/ μ l and pRL-TK at 5 ng/ μ l, together with the p300 (C-20:585) or CBP (C-20:583) antibodies, or rabbit IgG as a control, each at 1 μ g/ μ l. The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (−NGF) for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =6. Bim -LUC is activated significantly following NGF withdrawal when co-injected with rabbit IgG ( P =0.0001). Bim -LUC is not activated significantly when co-microinjected with the p300 and CBP antibodies. There is a significant decrease in the induction of bim -LUC following NGF withdrawal when co-injected with the p300 antibody ( P =0.034) and the CBP antibody ( P =0.013) compared with the control rabbit IgG. (b) Sympathetic neurons were microinjected with pCRE-LUC or pLUC (as a control), each at 20 ng/ μ l, and pRL-TK at 5 ng/ μ l, together with the p300 (C-20:585) or CBP (C-20:583) antibodies or rabbit IgG, each at 1 μ g/ μ l. After injection, the cells were treated with 500 μ CPTcAMP for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =5. The pCRE-LUC is activated significantly following addition of CPTcAMP, in comparison with pLUC ( P =0.0002). There is a significant decrease in the activation of pCRE-LUC following addition of CPTcAMP when co-injected with the p300 antibody ( P =0.019) and the CBP antibody ( P =0.028) compared with the control rabbit IgG. ( c ) Co-microinjection of the p300 expression vector, CMVp300, or the CBP expression vector, pRc/RSV-mCBP, with bim -LUC. Sympathetic neurons were microinjected with bim -LUC at 10 ng/ μ l and pRL-TK at 5 ng/ μ l, along with CMVp300 or the control empty vector pcDNA3.1 at 50 ng/ μ l, or pRc/RSV-mCBP or the control empty vector pBJ9Ω at 50 ng/ μ l. Following injection the cells were maintained in medium containing NGF for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =4. Co-microinjection of CMVp300 or pRc/RSV-mCBP induces a significant activation of bim -LUC ( P =0.016 and P =0.049, respectively). ( d ) The ICB is required for bim activation by p300. Sympathetic neurons were microinjected with bim -LUC or bim -LUC CCAAT (−) at 10 ng/ μ l and pRL-TK at 5 ng/ μ l, along with CMVp300 or the control empty vector pcDNA3.1 at 50 ng/ μ l. Following injection, the cells were maintained in medium containing NGF for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =4. Co-microinjection of CMVp300 induces a significant activation of bim -LUC ( P =0.013). Co-microinjection of CMVp300 does not activate bim -LUC CCAAT (−). ( *** P

    Journal: Cell Death and Differentiation

    Article Title: NF-Y is essential for expression of the proapoptotic bim gene in sympathetic neurons

    doi: 10.1038/cdd.2010.166

    Figure Lengend Snippet: CBP and p300 are important for activation of the bim promoter in sympathetic neurons. ( a ) Co-microinjection of antibodies specific to p300 or CBP with bim -LUC. Sympathetic neurons were microinjected with bim -LUC at 10 ng/ μ l and pRL-TK at 5 ng/ μ l, together with the p300 (C-20:585) or CBP (C-20:583) antibodies, or rabbit IgG as a control, each at 1 μ g/ μ l. The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (−NGF) for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =6. Bim -LUC is activated significantly following NGF withdrawal when co-injected with rabbit IgG ( P =0.0001). Bim -LUC is not activated significantly when co-microinjected with the p300 and CBP antibodies. There is a significant decrease in the induction of bim -LUC following NGF withdrawal when co-injected with the p300 antibody ( P =0.034) and the CBP antibody ( P =0.013) compared with the control rabbit IgG. (b) Sympathetic neurons were microinjected with pCRE-LUC or pLUC (as a control), each at 20 ng/ μ l, and pRL-TK at 5 ng/ μ l, together with the p300 (C-20:585) or CBP (C-20:583) antibodies or rabbit IgG, each at 1 μ g/ μ l. After injection, the cells were treated with 500 μ CPTcAMP for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =5. The pCRE-LUC is activated significantly following addition of CPTcAMP, in comparison with pLUC ( P =0.0002). There is a significant decrease in the activation of pCRE-LUC following addition of CPTcAMP when co-injected with the p300 antibody ( P =0.019) and the CBP antibody ( P =0.028) compared with the control rabbit IgG. ( c ) Co-microinjection of the p300 expression vector, CMVp300, or the CBP expression vector, pRc/RSV-mCBP, with bim -LUC. Sympathetic neurons were microinjected with bim -LUC at 10 ng/ μ l and pRL-TK at 5 ng/ μ l, along with CMVp300 or the control empty vector pcDNA3.1 at 50 ng/ μ l, or pRc/RSV-mCBP or the control empty vector pBJ9Ω at 50 ng/ μ l. Following injection the cells were maintained in medium containing NGF for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =4. Co-microinjection of CMVp300 or pRc/RSV-mCBP induces a significant activation of bim -LUC ( P =0.016 and P =0.049, respectively). ( d ) The ICB is required for bim activation by p300. Sympathetic neurons were microinjected with bim -LUC or bim -LUC CCAAT (−) at 10 ng/ μ l and pRL-TK at 5 ng/ μ l, along with CMVp300 or the control empty vector pcDNA3.1 at 50 ng/ μ l. Following injection, the cells were maintained in medium containing NGF for 16 h, after which time luciferase activity was measured. The data are presented as the mean±S.E., n =4. Co-microinjection of CMVp300 induces a significant activation of bim -LUC ( P =0.013). Co-microinjection of CMVp300 does not activate bim -LUC CCAAT (−). ( *** P

    Article Snippet: Secondary antibodies were a FITC-conjugated anti-rabbit IgG, and a rhodamine anti-gp IgG if neurons were microinjected (both from Jackson ImmunoResearch).

    Techniques: Activation Assay, Luciferase, Activity Assay, Injection, Expressing, Plasmid Preparation

    Caspase-4 interacts by cathepsin G by Td92 treatment. ( a ) HGFs were treated with Td92 (10 μ g/ml) or Td-G (5 μ g/ml) for 1 h. Stimulated cells were incubated with anti-cathepsin G antibody and anti-caspase-4 antibody followed by incubation with Cy3- and FITC-conjugated secondary antibodies, respectively. Cells were imaged by confocal laser scanning microscopy. Scale bars represent 50 μ m. ( b ) HGFs were pretreated with cathepsin G inhibitor I (CatG I) for 30 min or transfected with control siRNA or siRNA specific for cathepsin G for 24 h prior to stimulation with Td92 for 1 h. Images of 10 microscopic fields per well were taken at × 200 magnification, and the percentage of coaggregates was calculated relative to total cells using NIH ImageJ software. At least 500 cells were counted for each experimental condition. Data are shown as the means±S.D. of three independent experiments. * P

    Journal: Cell Death and Differentiation

    Article Title: Caspase-4 activation by a bacterial surface protein is mediated by cathepsin G in human gingival fibroblasts

    doi: 10.1038/cdd.2017.167

    Figure Lengend Snippet: Caspase-4 interacts by cathepsin G by Td92 treatment. ( a ) HGFs were treated with Td92 (10 μ g/ml) or Td-G (5 μ g/ml) for 1 h. Stimulated cells were incubated with anti-cathepsin G antibody and anti-caspase-4 antibody followed by incubation with Cy3- and FITC-conjugated secondary antibodies, respectively. Cells were imaged by confocal laser scanning microscopy. Scale bars represent 50 μ m. ( b ) HGFs were pretreated with cathepsin G inhibitor I (CatG I) for 30 min or transfected with control siRNA or siRNA specific for cathepsin G for 24 h prior to stimulation with Td92 for 1 h. Images of 10 microscopic fields per well were taken at × 200 magnification, and the percentage of coaggregates was calculated relative to total cells using NIH ImageJ software. At least 500 cells were counted for each experimental condition. Data are shown as the means±S.D. of three independent experiments. * P

    Article Snippet: Cells were then incubated with rabbit anti-cathepsin G Ab, goat anti-caspase-4 Ab, mouse ant-LAMP-1 Ab, isotype rabbit IgG, isotype goat IgG or isotype mouse IgG followed by incubation with Cy3- and FITC-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, Baltimore, PA, USA) in blocking solution.

    Techniques: Incubation, Confocal Laser Scanning Microscopy, Transfection, Software

    mpc constitutively express 5 monocyte chemotactic factors. (A) RT-PCR analysis of mpc mRNA at day 14 of FKN, MDC, MCP-1, VEGF, and β2M (β2microglobulin). (B and C) ELISA for (B) MDC, MCP-1, and (C) VEGF of mpc supernatants. Each symbol represents one culture estimated in triplicate. (D–G) Immunolabeling of (D) FKN, (E) MDC, (F) MCP-1, (G) VEGF using FITC-conjugated secondary antibody. Blue, DAPI stain.

    Journal: The Journal of Cell Biology

    Article Title: Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth

    doi: 10.1083/jcb.200212046

    Figure Lengend Snippet: mpc constitutively express 5 monocyte chemotactic factors. (A) RT-PCR analysis of mpc mRNA at day 14 of FKN, MDC, MCP-1, VEGF, and β2M (β2microglobulin). (B and C) ELISA for (B) MDC, MCP-1, and (C) VEGF of mpc supernatants. Each symbol represents one culture estimated in triplicate. (D–G) Immunolabeling of (D) FKN, (E) MDC, (F) MCP-1, (G) VEGF using FITC-conjugated secondary antibody. Blue, DAPI stain.

    Article Snippet: mpc labelings mpc were labeled with primary antibodies for 2 h: 10 μg/ml anti-MCP1, 50 μg/ml anti-FKN,10 μg/ml anti-MDC, 10 μg/ml anti-VEGF, revealed using FITC-conjugated secondary antibody (1:100 dilution; Jackson ImmunoResearch Laboratories) or biotin-conjugated secondary antibody (1:150 dilution; Jackson ImmunoResearch Laboratories) and FITC-streptavidin (1:50 dilution; Vector Laboratories).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunolabeling, Staining

    Subtypes of VSMCs determine expression levels of PDI and proliferation or apoptosis of VSMCs induced by SS, AGEs or both. The quiescent cultured VSMCs were subjected to SS and/or AGEs for 1 h, and continually cultured for additional 24 h. These cells were then stained with primary PDI and Ki-67 antibodies, FITC or Cy3-conjugated secondary antibodies, and a TUNEL kit, and then counterstained with DAPI. ( a–d ) The triple-labeling immunofluorescent shows Ki-67-positive (red), and PDI-labeling cells (green). ( e–h ) TUNEL-positive (green) and PDI-labeling cells (red). ( i–l ) SM- α -actin positive (green) and PDI-positive cells (red). ( m–p ) SM- α -actin positive (green) and Ki67-positive cells (red); ( q–t ) SM- α -actin positive (red) and TUNEL-positive cells (green). Using ImageJ program, the cells were sorted into two groups, cells with low and negative PDI or SM- α -actin expression and high and strong PDI or SM- α -actin expression. Then the Ki67-positive or TUNEL-positive cells were counted, respectively. DAPI stained cells were used for total cell numbers. ( u and v ) Statistical results of the ratio of Ki-67- or TUNEL-positive cells and PDI from ( a to d ) and ( e to h, ) respectively; ( w and y ) show statistical results of the ratio of Ki-67- or TUNEL-positive cells and SM- α -actin from ( m to p ) and ( q to t ), respectively; ( x ) shows statistical results of the ratio of PDI and SM- α -actin from ( i to l ). These statistical results were obtained from three independent experiments. a, P

    Journal: Cell Death & Disease

    Article Title: Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis

    doi: 10.1038/cddis.2017.213

    Figure Lengend Snippet: Subtypes of VSMCs determine expression levels of PDI and proliferation or apoptosis of VSMCs induced by SS, AGEs or both. The quiescent cultured VSMCs were subjected to SS and/or AGEs for 1 h, and continually cultured for additional 24 h. These cells were then stained with primary PDI and Ki-67 antibodies, FITC or Cy3-conjugated secondary antibodies, and a TUNEL kit, and then counterstained with DAPI. ( a–d ) The triple-labeling immunofluorescent shows Ki-67-positive (red), and PDI-labeling cells (green). ( e–h ) TUNEL-positive (green) and PDI-labeling cells (red). ( i–l ) SM- α -actin positive (green) and PDI-positive cells (red). ( m–p ) SM- α -actin positive (green) and Ki67-positive cells (red); ( q–t ) SM- α -actin positive (red) and TUNEL-positive cells (green). Using ImageJ program, the cells were sorted into two groups, cells with low and negative PDI or SM- α -actin expression and high and strong PDI or SM- α -actin expression. Then the Ki67-positive or TUNEL-positive cells were counted, respectively. DAPI stained cells were used for total cell numbers. ( u and v ) Statistical results of the ratio of Ki-67- or TUNEL-positive cells and PDI from ( a to d ) and ( e to h, ) respectively; ( w and y ) show statistical results of the ratio of Ki-67- or TUNEL-positive cells and SM- α -actin from ( m to p ) and ( q to t ), respectively; ( x ) shows statistical results of the ratio of PDI and SM- α -actin from ( i to l ). These statistical results were obtained from three independent experiments. a, P

    Article Snippet: The sections were incubated with primary antibodies, Ki67, and PDI antibodies (1:200, Santa Cruz, CA, USA), SMC-α -actin antibody (1:600, Sigma) mixed in 0.3% Triton X-100 overnight at 4 °C, followed by incubation with CY3- and FITC-conjugated secondary antibodies (1:200, Jackson Immuno Research Laboratories Inc, West Grove, PA, USA) for 2 h at 37 °C.

    Techniques: Expressing, Cell Culture, Staining, TUNEL Assay, Labeling

    PDI upregulation in smooth muscle cells leads to accelerated diabetic vein graft arteriosclerosis. Paraffin-embedded sections of the vein grafts of non-diabetic and diabetic mice killed 8 weeks after surgery were used for: ( a , b ) H E staining shows the wall structures in vein grafts of non-diabetic and diabetic mice and the respective vena cava (A’, B’) were used for the control of the vein grafts; ( c, d ) Masson trichromatic staining shows the wall structures and deposited collagenous fibers (blue color) in the vein grafts of non-diabetic and diabetic mice; ( e–l ) triple-labeling immunofluorescence with primary PDI, SM-alpha-actin antibodies and FITC, or Cy3-conjugated secondary antibody was performed and counterstained with DAPI; ( e, f ) DAPI staining shows cell nuclei (blue) in the walls of vein grafts from non-diabetic and diabetic mice; ( g, h ) SM- α -actin positive cells (green) show VSMCs in the vein grafts from non-diabetic and diabetic mice; ( i, j ) the cells with PDI expression (red) in the vein grafts from non-diabetic and diabetic mice; ( k, l ) merged images show co-expression (yellow) of PDI (red) and SM-alpha-actin (green) in VSMCs in the walls of vein grafts from ( e to j ). ( m ) Statistical results show ratios of PDI-positive cell numbers/ total cell numbers from ( e, f and i, j ) from three independent experiments; ( n ) statistical results show wall thickness of vein grafts from ( a, b ) from third independent experiments. a , P

    Journal: Cell Death & Disease

    Article Title: Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis

    doi: 10.1038/cddis.2017.213

    Figure Lengend Snippet: PDI upregulation in smooth muscle cells leads to accelerated diabetic vein graft arteriosclerosis. Paraffin-embedded sections of the vein grafts of non-diabetic and diabetic mice killed 8 weeks after surgery were used for: ( a , b ) H E staining shows the wall structures in vein grafts of non-diabetic and diabetic mice and the respective vena cava (A’, B’) were used for the control of the vein grafts; ( c, d ) Masson trichromatic staining shows the wall structures and deposited collagenous fibers (blue color) in the vein grafts of non-diabetic and diabetic mice; ( e–l ) triple-labeling immunofluorescence with primary PDI, SM-alpha-actin antibodies and FITC, or Cy3-conjugated secondary antibody was performed and counterstained with DAPI; ( e, f ) DAPI staining shows cell nuclei (blue) in the walls of vein grafts from non-diabetic and diabetic mice; ( g, h ) SM- α -actin positive cells (green) show VSMCs in the vein grafts from non-diabetic and diabetic mice; ( i, j ) the cells with PDI expression (red) in the vein grafts from non-diabetic and diabetic mice; ( k, l ) merged images show co-expression (yellow) of PDI (red) and SM-alpha-actin (green) in VSMCs in the walls of vein grafts from ( e to j ). ( m ) Statistical results show ratios of PDI-positive cell numbers/ total cell numbers from ( e, f and i, j ) from three independent experiments; ( n ) statistical results show wall thickness of vein grafts from ( a, b ) from third independent experiments. a , P

    Article Snippet: The sections were incubated with primary antibodies, Ki67, and PDI antibodies (1:200, Santa Cruz, CA, USA), SMC-α -actin antibody (1:600, Sigma) mixed in 0.3% Triton X-100 overnight at 4 °C, followed by incubation with CY3- and FITC-conjugated secondary antibodies (1:200, Jackson Immuno Research Laboratories Inc, West Grove, PA, USA) for 2 h at 37 °C.

    Techniques: Mouse Assay, Staining, Labeling, Immunofluorescence, Expressing