biotin labelled antihuman igg1 igg2 igg3 igg4  (Millipore)


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    Structured Review

    Millipore biotin labelled antihuman igg1 igg2 igg3 igg4
    Biotin Labelled Antihuman Igg1 Igg2 Igg3 Igg4, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotin labelled antihuman igg1 igg2 igg3 igg4/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotin labelled antihuman igg1 igg2 igg3 igg4 - by Bioz Stars, 2020-07
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    peroxidase-labelled antihuman igg

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    Incubation:

    Article Title: Autoantibodies Against β1‐Adrenoceptor Exaggerated Ventricular Remodeling by Inhibiting CTRP9 Expression
    Article Snippet: .. After washing 3 times, biotinylated goat‐antihuman IgG antibodies (Sigma) at 1:1000 dilution in PMT were added and incubated for 1 hour at 37°C. .. After washing 3 times, streptavidin‐peroxidase conjugate (Sigma) at 1:2000 dilution in the same buffer was added to the wells and incubated under the same conditions.

    other:

    Article Title: ?1-Adrenoceptor Autoantibodies from DCM Patients Enhance the Proliferation of T Lymphocytes through the ?1-AR/cAMP/PKA and p38 MAPK Pathways
    Article Snippet: After 3 washings, biotinylated goat-antihuman IgG antibodies (Sigma) (1∶1000 dilutions in PMT) were added for 1 h at 37°C.

    Article Title: Detection of Trichomonas vaginalis in prostate tissue and serostatus in patients with asymptomatic benign prostatic hyperplasia
    Article Snippet: The horseradish peroxidase-labelled antihuman IgG and the biotin-labelled antihuman IgG1/IgG2/IgG3/IgG4 (Sigma, St. Louis, MO, USA) were used at the calculated optimum dilution in PBS—Tween 20—BSA.

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  • 92
    Millipore goat anti human igg
    RTS,S vaccine-induced immunity declines over time. A random selection of children vaccinated with RTS,S (Manhiça cohort, n = 30) was tested for C1q-fixation ( a ), <t>IgG/IgM</t> ( b ), and IgG subclasses ( c ) to CSP at months 3, 8.5, 21, 33, 45, and 6. Note that due to low reactivity, C1q-fixation was re-tested at a higher dilution of 1/110, in addition to 1/250, to confirm results. Samples were tested in duplicate, and the median and 95% CI of the median from each time point group are shown by the symbol and shaded area, respectively
    Goat Anti Human Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti human igg/product/Millipore
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    goat anti human igg - by Bioz Stars, 2020-07
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    93
    Millipore cy3 conjugated anti rabbit igg
    Localization of FGFRs in human sperm. Sperm cells were stained with anti FGFR1, FGFR2, FGFR3 and FGFR4 or rabbit <t>IgG</t> and a secondary antibody labeled with <t>Cy3.</t> The corresponding fields stained with FITC-PSA to assess acrosomal status are shown. Bar: 10 μm. On the right, a representative image of individual sperm is depicted; ( A ) sperm stained with anti FGFR antibody and Cy3-conjugated secondary antibody, ( B ) FITC-PSA, ( C ) merge.
    Cy3 Conjugated Anti Rabbit Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy3 conjugated anti rabbit igg/product/Millipore
    Average 93 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
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    92
    Millipore olig2
    No significant change in Pax2 + interneuron progenitors in the <t>Olig2</t> -null cerebellum. ( A ) Immunnostaining analysis is performed on mid-sagittal cerebellar sections of the E18.5 Olig2 -null mice. The number of Pax2 + cells per cerebellar section is quantified and compared. No significant change in the number of Pax2 + interneuron progenitors is detected between Olig2 -null and control mice (n = 3 for both genotypes). ( B ) Similar immunostaining was also performed and quantified on cerebellar sections of E18.5 Olig1 -null mice. DAPI is used to label nuclei. Scale bar: 200 μm. n.s., not significant by Student’s t -test.
    Olig2, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/olig2/product/Millipore
    Average 92 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    olig2 - by Bioz Stars, 2020-07
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    91
    Millipore control igg antibody
    Robo1 signaling activates Rho GTPases in transformed cells. Src transformed cells were treated with <t>R5</t> antibody or control antiserum <t>(IgG)</t> and examined for total and activated Cdc42 and Rac1 GTPases in panels a and b , respectively. Western blotting was performed to detect active (GTP bound) Cdc42 and Rac1, total Cdc42 and Rac1, and GST. Levels of active Cdc42 and Rac1 were quantitated and shown as the percent of untreated control cells (mean+SEM, n=3). Experiments were performed with Cx43Ko and wild type cells, with results from Cx43Ko cells shown. Single and triple asterisks indicate p values less than 0.5 and 0.005, respectively (by t-test).
    Control Igg Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/control igg antibody/product/Millipore
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    control igg antibody - by Bioz Stars, 2020-07
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    Image Search Results


    RTS,S vaccine-induced immunity declines over time. A random selection of children vaccinated with RTS,S (Manhiça cohort, n = 30) was tested for C1q-fixation ( a ), IgG/IgM ( b ), and IgG subclasses ( c ) to CSP at months 3, 8.5, 21, 33, 45, and 6. Note that due to low reactivity, C1q-fixation was re-tested at a higher dilution of 1/110, in addition to 1/250, to confirm results. Samples were tested in duplicate, and the median and 95% CI of the median from each time point group are shown by the symbol and shaded area, respectively

    Journal: BMC Medicine

    Article Title: Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure

    doi: 10.1186/s12916-019-1277-x

    Figure Lengend Snippet: RTS,S vaccine-induced immunity declines over time. A random selection of children vaccinated with RTS,S (Manhiça cohort, n = 30) was tested for C1q-fixation ( a ), IgG/IgM ( b ), and IgG subclasses ( c ) to CSP at months 3, 8.5, 21, 33, 45, and 6. Note that due to low reactivity, C1q-fixation was re-tested at a higher dilution of 1/110, in addition to 1/250, to confirm results. Samples were tested in duplicate, and the median and 95% CI of the median from each time point group are shown by the symbol and shaded area, respectively

    Article Snippet: Plates were washed, and antibody isotypes were detected using goat anti-human IgG and IgM conjugated to horseradish peroxidase (HRP, Millipore, Burlington, USA) at 1/2500 dilution in buffer for 1 h at RT.

    Techniques: Selection

    RTS,S vaccine-induced IgG and IgM antibodies to CSP. Children in RTS,S and comparator vaccine groups from Manhiça (black box plots; N = 50 and N = 25, respectively) and Ilha Josina cohorts (gray box plots; N = 49 and N = 24, respectively) were tested for IgG ( a ), IgG subclasses ( b ), and IgM ( c ) to CSP. Sera collected at baseline (month 0, M0) and after vaccination (month 3, M3) were tested in duplicate (note that only M3 was tested in b ), and the mean value was used to generate box plots whereby top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff value (dashed lines), and the percentage of individuals above this threshold are shown. Reactivity between paired samples and unpaired samples were compared using Wilcoxon matched-pairs signed-rank test and Mann-Whitney U test, respectively

    Journal: BMC Medicine

    Article Title: Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure

    doi: 10.1186/s12916-019-1277-x

    Figure Lengend Snippet: RTS,S vaccine-induced IgG and IgM antibodies to CSP. Children in RTS,S and comparator vaccine groups from Manhiça (black box plots; N = 50 and N = 25, respectively) and Ilha Josina cohorts (gray box plots; N = 49 and N = 24, respectively) were tested for IgG ( a ), IgG subclasses ( b ), and IgM ( c ) to CSP. Sera collected at baseline (month 0, M0) and after vaccination (month 3, M3) were tested in duplicate (note that only M3 was tested in b ), and the mean value was used to generate box plots whereby top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff value (dashed lines), and the percentage of individuals above this threshold are shown. Reactivity between paired samples and unpaired samples were compared using Wilcoxon matched-pairs signed-rank test and Mann-Whitney U test, respectively

    Article Snippet: Plates were washed, and antibody isotypes were detected using goat anti-human IgG and IgM conjugated to horseradish peroxidase (HRP, Millipore, Burlington, USA) at 1/2500 dilution in buffer for 1 h at RT.

    Techniques: MANN-WHITNEY

    High variability among RTS,S vaccine-induced IgG targeting the central repeat and C-terminal regions of CSP. Children in RTS,S vaccine group from Manhiça and Ilha Josina cohorts ( N = 99, 3 M) were tested for IgG to NANP and CT and C1q-fixation to CSP, and the values were used for the following analysis. a Heat map of children arranged in descending (top to bottom) order of C1q-fixation (left), corresponding IgG to NANP and CT (middle), and for comparison IgM to NANP and CT (right). b Variability between epitope specificity was quantified by calculating the ratio of NANP-to-CT IgG. Children with low variability were considered to have equal reactivity to NANP and CT (ratio between 0.75 and 1.25 shown in white), and children exceeding this range were considered to have a NANP- or CT-skewed response (ratio > 1.25 shown in purple and ratio

    Journal: BMC Medicine

    Article Title: Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure

    doi: 10.1186/s12916-019-1277-x

    Figure Lengend Snippet: High variability among RTS,S vaccine-induced IgG targeting the central repeat and C-terminal regions of CSP. Children in RTS,S vaccine group from Manhiça and Ilha Josina cohorts ( N = 99, 3 M) were tested for IgG to NANP and CT and C1q-fixation to CSP, and the values were used for the following analysis. a Heat map of children arranged in descending (top to bottom) order of C1q-fixation (left), corresponding IgG to NANP and CT (middle), and for comparison IgM to NANP and CT (right). b Variability between epitope specificity was quantified by calculating the ratio of NANP-to-CT IgG. Children with low variability were considered to have equal reactivity to NANP and CT (ratio between 0.75 and 1.25 shown in white), and children exceeding this range were considered to have a NANP- or CT-skewed response (ratio > 1.25 shown in purple and ratio

    Article Snippet: Plates were washed, and antibody isotypes were detected using goat anti-human IgG and IgM conjugated to horseradish peroxidase (HRP, Millipore, Burlington, USA) at 1/2500 dilution in buffer for 1 h at RT.

    Techniques:

    Functional complement-fixing antibodies target the central repeat and C-terminal regions of CSP. Children in RTS,S vaccine group from Manhiça (black box plots; N = 50) and Ilha Josina cohorts (gray box plots; N = 49) were tested for IgG ( a ) and IgM ( b ) to NANP and CT regions of CSP. Sera collected at baseline (month 0, M0) and after vaccination (month 3, M3) were tested in duplicate, and the mean value was used to generate box plots whereby top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff values (dashed lines), and the percentages of individuals above this threshold are shown. Reactivity between paired samples was compared using Wilcoxon matched-pairs signed-rank test. c , d Children in RTS,S vaccine group from Manhiça and Ilha Josina cohorts ( N = 99, M3) were tested for C1q-fixation to CSP, NANP, and CT, and the values were plotted compared to IgG reactivity (c) and C1q fixation to CSP, NANP, and CT were correlated (d)

    Journal: BMC Medicine

    Article Title: Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure

    doi: 10.1186/s12916-019-1277-x

    Figure Lengend Snippet: Functional complement-fixing antibodies target the central repeat and C-terminal regions of CSP. Children in RTS,S vaccine group from Manhiça (black box plots; N = 50) and Ilha Josina cohorts (gray box plots; N = 49) were tested for IgG ( a ) and IgM ( b ) to NANP and CT regions of CSP. Sera collected at baseline (month 0, M0) and after vaccination (month 3, M3) were tested in duplicate, and the mean value was used to generate box plots whereby top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff values (dashed lines), and the percentages of individuals above this threshold are shown. Reactivity between paired samples was compared using Wilcoxon matched-pairs signed-rank test. c , d Children in RTS,S vaccine group from Manhiça and Ilha Josina cohorts ( N = 99, M3) were tested for C1q-fixation to CSP, NANP, and CT, and the values were plotted compared to IgG reactivity (c) and C1q fixation to CSP, NANP, and CT were correlated (d)

    Article Snippet: Plates were washed, and antibody isotypes were detected using goat anti-human IgG and IgM conjugated to horseradish peroxidase (HRP, Millipore, Burlington, USA) at 1/2500 dilution in buffer for 1 h at RT.

    Techniques: Functional Assay

    Localization of FGFRs in human sperm. Sperm cells were stained with anti FGFR1, FGFR2, FGFR3 and FGFR4 or rabbit IgG and a secondary antibody labeled with Cy3. The corresponding fields stained with FITC-PSA to assess acrosomal status are shown. Bar: 10 μm. On the right, a representative image of individual sperm is depicted; ( A ) sperm stained with anti FGFR antibody and Cy3-conjugated secondary antibody, ( B ) FITC-PSA, ( C ) merge.

    Journal: PLoS ONE

    Article Title: Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation

    doi: 10.1371/journal.pone.0127297

    Figure Lengend Snippet: Localization of FGFRs in human sperm. Sperm cells were stained with anti FGFR1, FGFR2, FGFR3 and FGFR4 or rabbit IgG and a secondary antibody labeled with Cy3. The corresponding fields stained with FITC-PSA to assess acrosomal status are shown. Bar: 10 μm. On the right, a representative image of individual sperm is depicted; ( A ) sperm stained with anti FGFR antibody and Cy3-conjugated secondary antibody, ( B ) FITC-PSA, ( C ) merge.

    Article Snippet: Other antibodies used were: anti pFGFR Tyr653/654 (#3476, Cell Signaling Technology, Inc., Beverley, MA, USA), anti pERK (sc-7383, Santa Cruz, and #4370, Cell Signaling), anti ERK (sc-94, Santa Cruz), anti pAkt Ser473 (sc-7985, Santa Cruz, and #4060, Cell Signaling), anti Akt (#4691, Cell Signaling), rabbit immunoglobulin G (IgG) (Sigma), horse-radish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma), Cy3-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG (Chemicon-Millipore, Billerica, MA, USA) and anti-rabbit IgG (Sigma).

    Techniques: Staining, Labeling

    No significant change in Pax2 + interneuron progenitors in the Olig2 -null cerebellum. ( A ) Immunnostaining analysis is performed on mid-sagittal cerebellar sections of the E18.5 Olig2 -null mice. The number of Pax2 + cells per cerebellar section is quantified and compared. No significant change in the number of Pax2 + interneuron progenitors is detected between Olig2 -null and control mice (n = 3 for both genotypes). ( B ) Similar immunostaining was also performed and quantified on cerebellar sections of E18.5 Olig1 -null mice. DAPI is used to label nuclei. Scale bar: 200 μm. n.s., not significant by Student’s t -test.

    Journal: Scientific Reports

    Article Title: Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum

    doi: 10.1038/srep30711

    Figure Lengend Snippet: No significant change in Pax2 + interneuron progenitors in the Olig2 -null cerebellum. ( A ) Immunnostaining analysis is performed on mid-sagittal cerebellar sections of the E18.5 Olig2 -null mice. The number of Pax2 + cells per cerebellar section is quantified and compared. No significant change in the number of Pax2 + interneuron progenitors is detected between Olig2 -null and control mice (n = 3 for both genotypes). ( B ) Similar immunostaining was also performed and quantified on cerebellar sections of E18.5 Olig1 -null mice. DAPI is used to label nuclei. Scale bar: 200 μm. n.s., not significant by Student’s t -test.

    Article Snippet: Tissue sections or fixed cell cultures were incubated with monoclonal antibodies against BrdU (rat IgG, 1:200, Accurate), Olig2 (mouse IgG, 1:200, Millipore), APC (mouse IgG, clone CC-1, 1:200, Calbiochem) and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against β-Galactosidase (β-gal) (chicken IgY, 1:2000, Abcam), BLBP (rabbit IgG, 1:500, Abcam), Tbr1 (rabbit IgG, 1:400, Proteintech), Pax2 (rabbit IgG, 1:50, Proteintech), Pax6 (rabbit IgG, 1:100, Proteintech), GABA (rabbit IgG, 1:2000, Sigma), Parvalbumin (rabbit IgG, 1:800, ImmunoStar), Olig2 (rabbit IgG, 1:500, Millipore or guinea pig IgG, 1:500, a gift from Dr. Ben Novitch at UCLA), GFAP (chicken IgY, 1:1000, Aves or rabbit IgG, 1:2000, DAKO), Calbindin (rabbit IgG, 1:500, ImmunoStar), Calretinin (rabbit IgG, 1:500, ImmunoStar), cleaved caspase-3 (rabbit IgG, 1:1000, Cell Signaling), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).

    Techniques: Mouse Assay, Immunostaining

    Olig2 -null mice show reduced PC generation. ( A ) Immunostaining with an anti-Calbindin antibody on E15.5 cerebellar mid-sagittal sections of Olig2 +/+ and Olig2 −/− mice shows notably reduction of PCs in the cerebellum (CB). Quantification on confocal images shows a significant reduction in the number of Calbindin + cells per cerebellar section in Olig2 −/− mice compared with controls (n = 3 for both genotypes). DAPI is used to label nuclei. MB, midbrain. The number of Calbindin + cells per field in the acute cultures of dissociated E17.5 Olig2 −/− ( B ) and E18.5 Olig1 −/− ( C ) cerebella are compared with their controls (n = 3 for both mutant and control genotypes). In these same acute cultures, the percentage of Pax6 + cells among total cells (DAPI + ) are also compared for E17.5 Olig2 −/− ( D ) and E18.5 Olig1 −/− ( E ) cerebella. Gene expression levels in dissected cerebellar tissues are compared by qRT-PCR between Olig2 +/− and Olig2 −/− mice (n = 4 for both genotypes) at E18.5 ( F ), or between Olig1 +/+,− and Olig1 −/− mice (n = 3 for both genotypes) at E17.5 ( G ). Scale bar: 200 μm in A and B ; 100 μm in D . * P

    Journal: Scientific Reports

    Article Title: Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum

    doi: 10.1038/srep30711

    Figure Lengend Snippet: Olig2 -null mice show reduced PC generation. ( A ) Immunostaining with an anti-Calbindin antibody on E15.5 cerebellar mid-sagittal sections of Olig2 +/+ and Olig2 −/− mice shows notably reduction of PCs in the cerebellum (CB). Quantification on confocal images shows a significant reduction in the number of Calbindin + cells per cerebellar section in Olig2 −/− mice compared with controls (n = 3 for both genotypes). DAPI is used to label nuclei. MB, midbrain. The number of Calbindin + cells per field in the acute cultures of dissociated E17.5 Olig2 −/− ( B ) and E18.5 Olig1 −/− ( C ) cerebella are compared with their controls (n = 3 for both mutant and control genotypes). In these same acute cultures, the percentage of Pax6 + cells among total cells (DAPI + ) are also compared for E17.5 Olig2 −/− ( D ) and E18.5 Olig1 −/− ( E ) cerebella. Gene expression levels in dissected cerebellar tissues are compared by qRT-PCR between Olig2 +/− and Olig2 −/− mice (n = 4 for both genotypes) at E18.5 ( F ), or between Olig1 +/+,− and Olig1 −/− mice (n = 3 for both genotypes) at E17.5 ( G ). Scale bar: 200 μm in A and B ; 100 μm in D . * P

    Article Snippet: Tissue sections or fixed cell cultures were incubated with monoclonal antibodies against BrdU (rat IgG, 1:200, Accurate), Olig2 (mouse IgG, 1:200, Millipore), APC (mouse IgG, clone CC-1, 1:200, Calbiochem) and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against β-Galactosidase (β-gal) (chicken IgY, 1:2000, Abcam), BLBP (rabbit IgG, 1:500, Abcam), Tbr1 (rabbit IgG, 1:400, Proteintech), Pax2 (rabbit IgG, 1:50, Proteintech), Pax6 (rabbit IgG, 1:100, Proteintech), GABA (rabbit IgG, 1:2000, Sigma), Parvalbumin (rabbit IgG, 1:800, ImmunoStar), Olig2 (rabbit IgG, 1:500, Millipore or guinea pig IgG, 1:500, a gift from Dr. Ben Novitch at UCLA), GFAP (chicken IgY, 1:1000, Aves or rabbit IgG, 1:2000, DAKO), Calbindin (rabbit IgG, 1:500, ImmunoStar), Calretinin (rabbit IgG, 1:500, ImmunoStar), cleaved caspase-3 (rabbit IgG, 1:1000, Cell Signaling), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).

    Techniques: Mouse Assay, Immunostaining, Mutagenesis, Expressing, Quantitative RT-PCR

    Differential expression patterns of Olig2 in the VZ progenitors relative to cell cycle in the E12.5 cerebellum and forebrain. Co-immunostainings were done on mid-sagittal sections of the cerebellum ( A ) and forebrain ( B ) of E12.5 embryos that have been pulse-labeled with BrdU for 30 min before sacrifice. ( A’,B’ ) are enlarged images of boxed regions in ( A , B ), respectively. The arrow indicates a double-positive cell in ( B’ ). ( C ) Comparison of BrdU labeling in the VZ Olig2 + cells between cerebellum and forebrain. Note that BrdU labels more than 50% of the Olig2 + cells (BrdU + /Olig2 + ) in the GE of the forebrain, but nearly none in the cerebellar VZ. ( D ) A series of BrdU-pulse labeling analyses of E12.5 cerebellar VZ progenitors. Percentages are shown to represent BrdU + and BrdU - fractions among Olig2 + cells in the cerebellar VZ. VZ, ventricular zone; GE, ganglionic eminence.

    Journal: Scientific Reports

    Article Title: Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum

    doi: 10.1038/srep30711

    Figure Lengend Snippet: Differential expression patterns of Olig2 in the VZ progenitors relative to cell cycle in the E12.5 cerebellum and forebrain. Co-immunostainings were done on mid-sagittal sections of the cerebellum ( A ) and forebrain ( B ) of E12.5 embryos that have been pulse-labeled with BrdU for 30 min before sacrifice. ( A’,B’ ) are enlarged images of boxed regions in ( A , B ), respectively. The arrow indicates a double-positive cell in ( B’ ). ( C ) Comparison of BrdU labeling in the VZ Olig2 + cells between cerebellum and forebrain. Note that BrdU labels more than 50% of the Olig2 + cells (BrdU + /Olig2 + ) in the GE of the forebrain, but nearly none in the cerebellar VZ. ( D ) A series of BrdU-pulse labeling analyses of E12.5 cerebellar VZ progenitors. Percentages are shown to represent BrdU + and BrdU - fractions among Olig2 + cells in the cerebellar VZ. VZ, ventricular zone; GE, ganglionic eminence.

    Article Snippet: Tissue sections or fixed cell cultures were incubated with monoclonal antibodies against BrdU (rat IgG, 1:200, Accurate), Olig2 (mouse IgG, 1:200, Millipore), APC (mouse IgG, clone CC-1, 1:200, Calbiochem) and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against β-Galactosidase (β-gal) (chicken IgY, 1:2000, Abcam), BLBP (rabbit IgG, 1:500, Abcam), Tbr1 (rabbit IgG, 1:400, Proteintech), Pax2 (rabbit IgG, 1:50, Proteintech), Pax6 (rabbit IgG, 1:100, Proteintech), GABA (rabbit IgG, 1:2000, Sigma), Parvalbumin (rabbit IgG, 1:800, ImmunoStar), Olig2 (rabbit IgG, 1:500, Millipore or guinea pig IgG, 1:500, a gift from Dr. Ben Novitch at UCLA), GFAP (chicken IgY, 1:1000, Aves or rabbit IgG, 1:2000, DAKO), Calbindin (rabbit IgG, 1:500, ImmunoStar), Calretinin (rabbit IgG, 1:500, ImmunoStar), cleaved caspase-3 (rabbit IgG, 1:1000, Cell Signaling), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).

    Techniques: Expressing, Labeling

    Olig2 -deletion induces impaired PC differentiation from cerebellar VZ progenitors without affecting proliferation. ( A ) Proliferation of cerebellar VZ progenitors at E12.5 is analyzed by their ability to incorporate BrdU in a 2-hour pulse-labeling treatment and compared between Olig2 +/+ and Olig2 −/− mice. ( B ) The percentage of BrdU + cells among total cells (DAPI + ) in the cerebellar VZ (as indicated) is calculated and compared between the two genotypes (n = 3 for both genotypes). ( C ) PC generation from the proliferating cerebellar VZ progenitors at E12.5 is also analyzed by co-staining of the postmitotic neuronal marker Lhx1/5 and BrdU in Olig2 +/+ and Olig2 −/− mice. ( D ) Percentage of Lhx1/5 + cells among BrdU + cells in the dorsal portion of the cerebellar VZ is quantified and compared between the two genotypes (n = 3 for both genotypes). VZ, ventricular zone. Scale bar: 20 μm in A and C . * P

    Journal: Scientific Reports

    Article Title: Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum

    doi: 10.1038/srep30711

    Figure Lengend Snippet: Olig2 -deletion induces impaired PC differentiation from cerebellar VZ progenitors without affecting proliferation. ( A ) Proliferation of cerebellar VZ progenitors at E12.5 is analyzed by their ability to incorporate BrdU in a 2-hour pulse-labeling treatment and compared between Olig2 +/+ and Olig2 −/− mice. ( B ) The percentage of BrdU + cells among total cells (DAPI + ) in the cerebellar VZ (as indicated) is calculated and compared between the two genotypes (n = 3 for both genotypes). ( C ) PC generation from the proliferating cerebellar VZ progenitors at E12.5 is also analyzed by co-staining of the postmitotic neuronal marker Lhx1/5 and BrdU in Olig2 +/+ and Olig2 −/− mice. ( D ) Percentage of Lhx1/5 + cells among BrdU + cells in the dorsal portion of the cerebellar VZ is quantified and compared between the two genotypes (n = 3 for both genotypes). VZ, ventricular zone. Scale bar: 20 μm in A and C . * P

    Article Snippet: Tissue sections or fixed cell cultures were incubated with monoclonal antibodies against BrdU (rat IgG, 1:200, Accurate), Olig2 (mouse IgG, 1:200, Millipore), APC (mouse IgG, clone CC-1, 1:200, Calbiochem) and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against β-Galactosidase (β-gal) (chicken IgY, 1:2000, Abcam), BLBP (rabbit IgG, 1:500, Abcam), Tbr1 (rabbit IgG, 1:400, Proteintech), Pax2 (rabbit IgG, 1:50, Proteintech), Pax6 (rabbit IgG, 1:100, Proteintech), GABA (rabbit IgG, 1:2000, Sigma), Parvalbumin (rabbit IgG, 1:800, ImmunoStar), Olig2 (rabbit IgG, 1:500, Millipore or guinea pig IgG, 1:500, a gift from Dr. Ben Novitch at UCLA), GFAP (chicken IgY, 1:1000, Aves or rabbit IgG, 1:2000, DAKO), Calbindin (rabbit IgG, 1:500, ImmunoStar), Calretinin (rabbit IgG, 1:500, ImmunoStar), cleaved caspase-3 (rabbit IgG, 1:1000, Cell Signaling), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).

    Techniques: Labeling, Mouse Assay, Staining, Marker

    Olig gene-expressing progenitors give rise to PCs and DCN neurons but rarely Pax2 + interneurons in the postnatal cerebellum as revealed by long-term lineage tracing. Olig1-Cre lineage tracing analysis is performed in the cerebellum of either Z/EG ( A–H ) reporter line at P18 or ROSA26;LacZ ( I ) reporter line at P16. Labeled cells with different morphology in the cerebellum of Olig1-Cre;Z/EG mice are revealed by their GFP expression ( A ). The identity of GFP + cells are examined by co-staining with cell-type specific markers Calbindin ( B ), Parvalbumin ( C ), GFAP ( D ), BLBP ( E ), Olig2 ( F,G ) and Pax2 ( H ). Some GFP + cells are indicated as either negative (arrows in C and H ) or positive (arrows in F,G ) for marker staining. The high magnification insert in ( C ) confirms the cellular identity of the indicated GFP + cell by showing the presence of DAPI + nucleus. Many labeled cells in the cerebellar DCN of Olig1-Cre;LacZ reporter mice are observed by anti-β-gal staining, some of which are NeuN + (arrow, I ) and GABA + (arrow, insert in I ). Olig2-Cre lineage tracing analysis is also performed in the cerebellum of ROSA26;tdTomato ( J–P ) reporter line at P22. Labeled cells with different morphology in the cerebellum of Olig2-Cre;tdTomato mice are revealed by their tdTomato expression ( J ). The identity of tdTomato + cells are examined by co-staining with cell-type specific markers Calbindin ( K ), Parvalbumin ( L ), GFAP ( M ), Olig2 ( N ), CC1 ( O ) and Pax2 ( P ). Note that two tdTomato + cells are co-labeled with Parvalbumin suggestive of small interneurons (arrows in L ). DAPI is used to label nuclei. ML, molecular layer; IGL, internal granule layer; WM, white matter; DCN, deep cerebellar nuclei. Scale bar: 100 μm in ( A,J ); 20 μm in ( B–H , K–P ); 25 μm in I .

    Journal: Scientific Reports

    Article Title: Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum

    doi: 10.1038/srep30711

    Figure Lengend Snippet: Olig gene-expressing progenitors give rise to PCs and DCN neurons but rarely Pax2 + interneurons in the postnatal cerebellum as revealed by long-term lineage tracing. Olig1-Cre lineage tracing analysis is performed in the cerebellum of either Z/EG ( A–H ) reporter line at P18 or ROSA26;LacZ ( I ) reporter line at P16. Labeled cells with different morphology in the cerebellum of Olig1-Cre;Z/EG mice are revealed by their GFP expression ( A ). The identity of GFP + cells are examined by co-staining with cell-type specific markers Calbindin ( B ), Parvalbumin ( C ), GFAP ( D ), BLBP ( E ), Olig2 ( F,G ) and Pax2 ( H ). Some GFP + cells are indicated as either negative (arrows in C and H ) or positive (arrows in F,G ) for marker staining. The high magnification insert in ( C ) confirms the cellular identity of the indicated GFP + cell by showing the presence of DAPI + nucleus. Many labeled cells in the cerebellar DCN of Olig1-Cre;LacZ reporter mice are observed by anti-β-gal staining, some of which are NeuN + (arrow, I ) and GABA + (arrow, insert in I ). Olig2-Cre lineage tracing analysis is also performed in the cerebellum of ROSA26;tdTomato ( J–P ) reporter line at P22. Labeled cells with different morphology in the cerebellum of Olig2-Cre;tdTomato mice are revealed by their tdTomato expression ( J ). The identity of tdTomato + cells are examined by co-staining with cell-type specific markers Calbindin ( K ), Parvalbumin ( L ), GFAP ( M ), Olig2 ( N ), CC1 ( O ) and Pax2 ( P ). Note that two tdTomato + cells are co-labeled with Parvalbumin suggestive of small interneurons (arrows in L ). DAPI is used to label nuclei. ML, molecular layer; IGL, internal granule layer; WM, white matter; DCN, deep cerebellar nuclei. Scale bar: 100 μm in ( A,J ); 20 μm in ( B–H , K–P ); 25 μm in I .

    Article Snippet: Tissue sections or fixed cell cultures were incubated with monoclonal antibodies against BrdU (rat IgG, 1:200, Accurate), Olig2 (mouse IgG, 1:200, Millipore), APC (mouse IgG, clone CC-1, 1:200, Calbiochem) and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against β-Galactosidase (β-gal) (chicken IgY, 1:2000, Abcam), BLBP (rabbit IgG, 1:500, Abcam), Tbr1 (rabbit IgG, 1:400, Proteintech), Pax2 (rabbit IgG, 1:50, Proteintech), Pax6 (rabbit IgG, 1:100, Proteintech), GABA (rabbit IgG, 1:2000, Sigma), Parvalbumin (rabbit IgG, 1:800, ImmunoStar), Olig2 (rabbit IgG, 1:500, Millipore or guinea pig IgG, 1:500, a gift from Dr. Ben Novitch at UCLA), GFAP (chicken IgY, 1:1000, Aves or rabbit IgG, 1:2000, DAKO), Calbindin (rabbit IgG, 1:500, ImmunoStar), Calretinin (rabbit IgG, 1:500, ImmunoStar), cleaved caspase-3 (rabbit IgG, 1:1000, Cell Signaling), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).

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

    Differential neuronal expression patterns of Olig2 + cells in the early embryonic cerebellum. Co-immunostaining is performed to analyze the expression of Olig2 ( A ) and a neuronal marker, DCX ( B ) on sagittal sections of the E12.5 cerebellum. The overlay image ( C ) reveals differential DCX expression patterns of the Olig2 + cells in the VZ and NTZ. The enlarged images of the boxed regions in ( C ) are shown in ( C1 ) and ( C2 ), respectively. Double-positive cells are pointed by arrows in ( C1 ) and an arrow in a higher power confocal image ( C1’ ). Double-positive cells (indicated by an arrow in C2’ , a higher power confocal image of C2 region) are very rarely seen in ( C2 ) where Olig2 and DCX show mostly a non-overlapping staining pattern. ( D ) Co-immunostaining of Olig2 and BrdU in the VZ of the E12.5 cerebellum that has been treated by a BrdU pulse-labeling. ( D’ ) The confocal image of the VZ in ( D ) showing a double-positive cell (arrow). ( E ) Co-expression analysis of Olig2 with markers of proliferation (Ki67), radial glia (BLBP) and PC differentiation (Lhx1/5) in the cerebellar VZ at E13.5. ( F ) A diagram depicting Olig2 expression range during the transition of VZ progenitors to early PCs. ( G ) Co-immunostaining with anti-Olig2 and anti-Pax2 on sagittal cerebellar sections of the E13.5 wild-type mouse. ( G’ ) is the enlarged image of boxed regions in ( G ) showing a non-overlapping staining pattern. VZ, ventricular zone; RL, rhombic lip; NTZ, nuclear transitory zone. Scale bar: 80 μm in A , B , C and G ; 25 μm in C1 and C2 ; 15 μm in C1’ and D’ ; 20 μm in C2’ , E and G’ ; 40 μm in D .

    Journal: Scientific Reports

    Article Title: Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum

    doi: 10.1038/srep30711

    Figure Lengend Snippet: Differential neuronal expression patterns of Olig2 + cells in the early embryonic cerebellum. Co-immunostaining is performed to analyze the expression of Olig2 ( A ) and a neuronal marker, DCX ( B ) on sagittal sections of the E12.5 cerebellum. The overlay image ( C ) reveals differential DCX expression patterns of the Olig2 + cells in the VZ and NTZ. The enlarged images of the boxed regions in ( C ) are shown in ( C1 ) and ( C2 ), respectively. Double-positive cells are pointed by arrows in ( C1 ) and an arrow in a higher power confocal image ( C1’ ). Double-positive cells (indicated by an arrow in C2’ , a higher power confocal image of C2 region) are very rarely seen in ( C2 ) where Olig2 and DCX show mostly a non-overlapping staining pattern. ( D ) Co-immunostaining of Olig2 and BrdU in the VZ of the E12.5 cerebellum that has been treated by a BrdU pulse-labeling. ( D’ ) The confocal image of the VZ in ( D ) showing a double-positive cell (arrow). ( E ) Co-expression analysis of Olig2 with markers of proliferation (Ki67), radial glia (BLBP) and PC differentiation (Lhx1/5) in the cerebellar VZ at E13.5. ( F ) A diagram depicting Olig2 expression range during the transition of VZ progenitors to early PCs. ( G ) Co-immunostaining with anti-Olig2 and anti-Pax2 on sagittal cerebellar sections of the E13.5 wild-type mouse. ( G’ ) is the enlarged image of boxed regions in ( G ) showing a non-overlapping staining pattern. VZ, ventricular zone; RL, rhombic lip; NTZ, nuclear transitory zone. Scale bar: 80 μm in A , B , C and G ; 25 μm in C1 and C2 ; 15 μm in C1’ and D’ ; 20 μm in C2’ , E and G’ ; 40 μm in D .

    Article Snippet: Tissue sections or fixed cell cultures were incubated with monoclonal antibodies against BrdU (rat IgG, 1:200, Accurate), Olig2 (mouse IgG, 1:200, Millipore), APC (mouse IgG, clone CC-1, 1:200, Calbiochem) and NeuN (mouse IgG, 1:400, Millipore); polyclonal antibodies against β-Galactosidase (β-gal) (chicken IgY, 1:2000, Abcam), BLBP (rabbit IgG, 1:500, Abcam), Tbr1 (rabbit IgG, 1:400, Proteintech), Pax2 (rabbit IgG, 1:50, Proteintech), Pax6 (rabbit IgG, 1:100, Proteintech), GABA (rabbit IgG, 1:2000, Sigma), Parvalbumin (rabbit IgG, 1:800, ImmunoStar), Olig2 (rabbit IgG, 1:500, Millipore or guinea pig IgG, 1:500, a gift from Dr. Ben Novitch at UCLA), GFAP (chicken IgY, 1:1000, Aves or rabbit IgG, 1:2000, DAKO), Calbindin (rabbit IgG, 1:500, ImmunoStar), Calretinin (rabbit IgG, 1:500, ImmunoStar), cleaved caspase-3 (rabbit IgG, 1:1000, Cell Signaling), and DCX (guinea pig IgG, 1:1000, Millipore), followed by appropriate species-specific secondary antibodies (Molecular Probes).

    Techniques: Expressing, Immunostaining, Marker, Staining, Labeling

    Robo1 signaling activates Rho GTPases in transformed cells. Src transformed cells were treated with R5 antibody or control antiserum (IgG) and examined for total and activated Cdc42 and Rac1 GTPases in panels a and b , respectively. Western blotting was performed to detect active (GTP bound) Cdc42 and Rac1, total Cdc42 and Rac1, and GST. Levels of active Cdc42 and Rac1 were quantitated and shown as the percent of untreated control cells (mean+SEM, n=3). Experiments were performed with Cx43Ko and wild type cells, with results from Cx43Ko cells shown. Single and triple asterisks indicate p values less than 0.5 and 0.005, respectively (by t-test).

    Journal: Oncotarget

    Article Title: Src activates Abl to augment Robo1 expression in order to promote tumor cell migration

    doi:

    Figure Lengend Snippet: Robo1 signaling activates Rho GTPases in transformed cells. Src transformed cells were treated with R5 antibody or control antiserum (IgG) and examined for total and activated Cdc42 and Rac1 GTPases in panels a and b , respectively. Western blotting was performed to detect active (GTP bound) Cdc42 and Rac1, total Cdc42 and Rac1, and GST. Levels of active Cdc42 and Rac1 were quantitated and shown as the percent of untreated control cells (mean+SEM, n=3). Experiments were performed with Cx43Ko and wild type cells, with results from Cx43Ko cells shown. Single and triple asterisks indicate p values less than 0.5 and 0.005, respectively (by t-test).

    Article Snippet: For some experiments, cells were treated overnight with 50 μg/ml cyclohexamide (Sigma, C7698), 60 nM R5 antibody to the extracellular region of Robo1, 60 nM control IgG antibody [ , ], or 40 μM GNF-2 Abl kinase inhibitor (Calbiochem, 197221).

    Techniques: Transformation Assay, Western Blot

    Src utilizes Robo1 to promote cell migration. Cells obtained from wild type (WT) or homozygous null Cx43 knockout (Cx43Ko) mouse embryos were transfected with v-Src or the empty parental vector and plated (10,000 per well) were plated on standard or ultra low attachment culture dishes to evaluate (a) anchored and (b) nonanchored cell growth. Cell numbers were determined by Coulter counter at the indicated time points for anchored cells or at 7 days for nonanchored cells. Data are shown as number of cells per well at the indicated time points (mean+SEM, n=3). (c) Cell migration was examined by a wound healing assay on nontransformed cells and Src transformed cells treated with IgG control antiserum, R5 antiserum to block Robo1 activity, or GNF-2 Abl kinase blocker. Migration was quantitated as the number of cells that entered a 1.8 mm2 area of the wound during 24 hours (mean+SEM, n=5). (d) Src transformed cells were treated with R5 antibody or control antiserum and analyzed by Western blotting for Robo1, active N-WASP (p-N-WASP), or β-actin. N-WASP activity was then quantitated and shown as percent of untreated control cells (mean+SEM, n=2). Experiments were performed with Cx43Ko and WT cells, with results from Cx43Ko cells shown in panel d. Double and triple asterisk indicate p values less than 0.01 and 0.005 compared to controls, respectively (by t-test).

    Journal: Oncotarget

    Article Title: Src activates Abl to augment Robo1 expression in order to promote tumor cell migration

    doi:

    Figure Lengend Snippet: Src utilizes Robo1 to promote cell migration. Cells obtained from wild type (WT) or homozygous null Cx43 knockout (Cx43Ko) mouse embryos were transfected with v-Src or the empty parental vector and plated (10,000 per well) were plated on standard or ultra low attachment culture dishes to evaluate (a) anchored and (b) nonanchored cell growth. Cell numbers were determined by Coulter counter at the indicated time points for anchored cells or at 7 days for nonanchored cells. Data are shown as number of cells per well at the indicated time points (mean+SEM, n=3). (c) Cell migration was examined by a wound healing assay on nontransformed cells and Src transformed cells treated with IgG control antiserum, R5 antiserum to block Robo1 activity, or GNF-2 Abl kinase blocker. Migration was quantitated as the number of cells that entered a 1.8 mm2 area of the wound during 24 hours (mean+SEM, n=5). (d) Src transformed cells were treated with R5 antibody or control antiserum and analyzed by Western blotting for Robo1, active N-WASP (p-N-WASP), or β-actin. N-WASP activity was then quantitated and shown as percent of untreated control cells (mean+SEM, n=2). Experiments were performed with Cx43Ko and WT cells, with results from Cx43Ko cells shown in panel d. Double and triple asterisk indicate p values less than 0.01 and 0.005 compared to controls, respectively (by t-test).

    Article Snippet: For some experiments, cells were treated overnight with 50 μg/ml cyclohexamide (Sigma, C7698), 60 nM R5 antibody to the extracellular region of Robo1, 60 nM control IgG antibody [ , ], or 40 μM GNF-2 Abl kinase inhibitor (Calbiochem, 197221).

    Techniques: Migration, Knock-Out, Transfection, Plasmid Preparation, Wound Healing Assay, Transformation Assay, Blocking Assay, Activity Assay, Western Blot