rabbit anti sox2  (Thermo Fisher)


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    anti SOX 2 Polyclonal Antibody
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    pa1-16968
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    Structured Review

    Thermo Fisher rabbit anti sox2
    Proposed model for bFGF signaling in pluripotent (green) vs. differentiated (orange) hiPSCs (for details see discussion). bFGF, basic fibroblast growth factor; OCT4, octamer-binding transcription factor 4; <t>SOX2,</t> sex determining region Y-box 2; GFAP, glial fibrillary acidic protein; RAS, rat sarcoma; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositidedependent protein kinase; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3

    https://www.bioz.com/result/rabbit anti sox2/product/Thermo Fisher
    Average 95 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    rabbit anti sox2 - by Bioz Stars, 2020-07
    95/100 stars

    Images

    1) Product Images from "bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling"

    Article Title: bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0307-1

    Proposed model for bFGF signaling in pluripotent (green) vs. differentiated (orange) hiPSCs (for details see discussion). bFGF, basic fibroblast growth factor; OCT4, octamer-binding transcription factor 4; SOX2, sex determining region Y-box 2; GFAP, glial fibrillary acidic protein; RAS, rat sarcoma; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositidedependent protein kinase; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3
    Figure Legend Snippet: Proposed model for bFGF signaling in pluripotent (green) vs. differentiated (orange) hiPSCs (for details see discussion). bFGF, basic fibroblast growth factor; OCT4, octamer-binding transcription factor 4; SOX2, sex determining region Y-box 2; GFAP, glial fibrillary acidic protein; RAS, rat sarcoma; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositidedependent protein kinase; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3

    Techniques Used: Binding Assay

    Long-term maintenance of undifferentiated hiPSCs. a Confocal imaging showed the expression of pluripotency markers (OCT4, SOX2, NANOG and SSEA4) and the absence of differentiation markers GFAP and α-SMA as ectodermal and mesodermal markers, respectively. Cell nuclei were stained with DAPI (blue). Scale bars, 10 μm. b Flow cytometry confirmed expression of OCT4, SSEA4 and SOX2 in hiPSCs with more than 98% of positive cells. c qPCR analysis for undifferentiated stem cell markers ( POU5F1 , SOX2 and NANOG ) and early commitment to differentiation markers ( BRACHYURY , PAX6 and AFP ). GAPDH was used as an internal control. d Immunoblot analysis showing the specificity of antibodies and expression of markers. HeLa cells were used as negative control, HFF and astrocytes were used as positive control for α-SMA and GFAP, respectively
    Figure Legend Snippet: Long-term maintenance of undifferentiated hiPSCs. a Confocal imaging showed the expression of pluripotency markers (OCT4, SOX2, NANOG and SSEA4) and the absence of differentiation markers GFAP and α-SMA as ectodermal and mesodermal markers, respectively. Cell nuclei were stained with DAPI (blue). Scale bars, 10 μm. b Flow cytometry confirmed expression of OCT4, SSEA4 and SOX2 in hiPSCs with more than 98% of positive cells. c qPCR analysis for undifferentiated stem cell markers ( POU5F1 , SOX2 and NANOG ) and early commitment to differentiation markers ( BRACHYURY , PAX6 and AFP ). GAPDH was used as an internal control. d Immunoblot analysis showing the specificity of antibodies and expression of markers. HeLa cells were used as negative control, HFF and astrocytes were used as positive control for α-SMA and GFAP, respectively

    Techniques Used: Imaging, Expressing, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    The critical role of bFGF for maintaining hiPSC pluripotency. a Phase contrast images of hiPSCs cultured under four different conditions, CM-100, CM-5, CM-0 and non-CM for 6 days. Undifferentiated hiPSCs formed compact colonies (CM-100), while without bFGF supplementation hiPSCs spread and flattened at day 6 (CM-0 and non-CM). Scale bar, 50 μm. b qPCR analysis showed the downregulation of pluripotency markers POU5F1 , SOX2 and NANOG in cells cultured in CM-0 and non-CM in comparison to control group (CM-100). All expression values were normalized to GAPDH . Results from three separate experiments, each carried out in triplicate, are shown as mean ± SD (ANOVA; * p
    Figure Legend Snippet: The critical role of bFGF for maintaining hiPSC pluripotency. a Phase contrast images of hiPSCs cultured under four different conditions, CM-100, CM-5, CM-0 and non-CM for 6 days. Undifferentiated hiPSCs formed compact colonies (CM-100), while without bFGF supplementation hiPSCs spread and flattened at day 6 (CM-0 and non-CM). Scale bar, 50 μm. b qPCR analysis showed the downregulation of pluripotency markers POU5F1 , SOX2 and NANOG in cells cultured in CM-0 and non-CM in comparison to control group (CM-100). All expression values were normalized to GAPDH . Results from three separate experiments, each carried out in triplicate, are shown as mean ± SD (ANOVA; * p

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    2) Product Images from "Genome Editing in Patient iPSCs Corrects the Most Prevalent USH2A Mutations and Reveals Intriguing Mutant mRNA Expression Profiles"

    Article Title: Genome Editing in Patient iPSCs Corrects the Most Prevalent USH2A Mutations and Reveals Intriguing Mutant mRNA Expression Profiles

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.11.016

    Genome Stability and Pluripotency of Corrected iPSC Lines (A and I) Phase-contrast images of USH2A -USH-iPSC clone B3B1 (A) and USH2A -RP-iPSC clone MS3F7 (I). (B and J) Genomic stability of USH2A -USH-iPSC B3B1 (B) and USH2A -RP-iPSC MS3F7 (J) as determined by a digital qPCR analysis of the most commonly rearranged regions reported in iPSCs. The copy number for each chromosomal position is shown with colored dots. (C–E and K–M) Pluripotency of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the markers OCT3/4 (C and K), SOX2 (D and L), and NANOG (E and M), respectively. Scale bars, 50 μM. (F-H and N-P) Differentiation capacity of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the germ layer markers GFAP (ectoderm; F and N), SMA (mesoderm; G and O), and AFP (endoderm; H and P), respectively. Scale bars, 20 μM.
    Figure Legend Snippet: Genome Stability and Pluripotency of Corrected iPSC Lines (A and I) Phase-contrast images of USH2A -USH-iPSC clone B3B1 (A) and USH2A -RP-iPSC clone MS3F7 (I). (B and J) Genomic stability of USH2A -USH-iPSC B3B1 (B) and USH2A -RP-iPSC MS3F7 (J) as determined by a digital qPCR analysis of the most commonly rearranged regions reported in iPSCs. The copy number for each chromosomal position is shown with colored dots. (C–E and K–M) Pluripotency of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the markers OCT3/4 (C and K), SOX2 (D and L), and NANOG (E and M), respectively. Scale bars, 50 μM. (F-H and N-P) Differentiation capacity of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the germ layer markers GFAP (ectoderm; F and N), SMA (mesoderm; G and O), and AFP (endoderm; H and P), respectively. Scale bars, 20 μM.

    Techniques Used: Real-time Polymerase Chain Reaction, Immunostaining

    3) Product Images from "Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation"

    Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08140-7

    Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p
    Figure Legend Snippet: Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p

    Techniques Used: Cell Culture, Staining

    Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p
    Figure Legend Snippet: Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p

    Techniques Used: In Vitro, Cell Culture, Staining, Marker, Expressing, Two Tailed Test

    4) Product Images from "Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation"

    Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08140-7

    Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p
    Figure Legend Snippet: Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p

    Techniques Used: Cell Culture, Staining

    Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p
    Figure Legend Snippet: Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p

    Techniques Used: In Vitro, Cell Culture, Staining, Marker, Expressing, Two Tailed Test

    5) Product Images from "Development of Circadian Oscillators in Neurosphere Cultures during Adult Neurogenesis"

    Article Title: Development of Circadian Oscillators in Neurosphere Cultures during Adult Neurogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122937

    The rhythmic state of spheres during early and late exposure to three culture conditions. A: Spheres were maintained in either SCM, SM or B27 medium. Spheres were imaged immediately after a forskolin treatment to synchronize circadian clock cells or after no treatment. Shown is the percentage of spheres that began in a particular state (C: circadian, U: ultradian, N: nonrhythmic) during the first 3 days of imaging (early) and their state during the final 3–4 days (late) of imaging sessions. Under differentiating conditions (SM or B27) the three paths to the circadian state (UC, CC, and NC) were most commonly observed. B: The increase in the percentage of spheres showing circadian rhythms is correlated with an increase in the differentiation marker Nestin - /GFAP + and negatively correlated with the decline in stem cell markers (Nestin + /GFAP + , Nestin + , and SOX2 + ) during 7 days in SM.
    Figure Legend Snippet: The rhythmic state of spheres during early and late exposure to three culture conditions. A: Spheres were maintained in either SCM, SM or B27 medium. Spheres were imaged immediately after a forskolin treatment to synchronize circadian clock cells or after no treatment. Shown is the percentage of spheres that began in a particular state (C: circadian, U: ultradian, N: nonrhythmic) during the first 3 days of imaging (early) and their state during the final 3–4 days (late) of imaging sessions. Under differentiating conditions (SM or B27) the three paths to the circadian state (UC, CC, and NC) were most commonly observed. B: The increase in the percentage of spheres showing circadian rhythms is correlated with an increase in the differentiation marker Nestin - /GFAP + and negatively correlated with the decline in stem cell markers (Nestin + /GFAP + , Nestin + , and SOX2 + ) during 7 days in SM.

    Techniques Used: Imaging, Marker

    Emergence of circadian rhythms before fully differentiated neurons appear. Spheres were synchronized by forskolin treatment and fixed after differentiation in SM or B27 medium, mimicking BLI conditions. Hoechst (blue) or propidium iodide (red) were used as nuclear stains. NSPCs were identified as SOX2 + (cyan; A - C : after 1, 4, 7 days in SM), Nestin + /GFAP + (yellow; D - F : after 1, 4, 7 days in SM; red: GFAP, green: Nestin) or Msi1 + (yellow; H : after 3 days in SM). Additional spheres were fixed after differentiation in medium with serum or B27 supplement to stain for progenitor cells as Dcx + (Yellow; G , J : after 4 days in SM or B27, respectively). Immature neuronal cells were identified as BetaIII-tubulin + (Green; K : after 5 days in B27 medium), and mature neuronal cells as NeuN + (Yellow; I : after 4 days in SM and Green; L : after 4 days in B27 medium). Scale bars = 50 μm, and A - C , E , H , I , and K are at the same magnification.
    Figure Legend Snippet: Emergence of circadian rhythms before fully differentiated neurons appear. Spheres were synchronized by forskolin treatment and fixed after differentiation in SM or B27 medium, mimicking BLI conditions. Hoechst (blue) or propidium iodide (red) were used as nuclear stains. NSPCs were identified as SOX2 + (cyan; A - C : after 1, 4, 7 days in SM), Nestin + /GFAP + (yellow; D - F : after 1, 4, 7 days in SM; red: GFAP, green: Nestin) or Msi1 + (yellow; H : after 3 days in SM). Additional spheres were fixed after differentiation in medium with serum or B27 supplement to stain for progenitor cells as Dcx + (Yellow; G , J : after 4 days in SM or B27, respectively). Immature neuronal cells were identified as BetaIII-tubulin + (Green; K : after 5 days in B27 medium), and mature neuronal cells as NeuN + (Yellow; I : after 4 days in SM and Green; L : after 4 days in B27 medium). Scale bars = 50 μm, and A - C , E , H , I , and K are at the same magnification.

    Techniques Used: Staining

    6) Product Images from "JAGGED1 IS NECESSARY FOR POSTNATAL AND ADULT NEUROGENESIS IN THE DENTATE GYRUS"

    Article Title: JAGGED1 IS NECESSARY FOR POSTNATAL AND ADULT NEUROGENESIS IN THE DENTATE GYRUS

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2014.02.004

    The transient increase in neurogenesis in the Nestin-CreER T2 ;Jagged1 F/F mice is accompanied by a reduction in the Nestin + and Sox2 + cell populations
    Figure Legend Snippet: The transient increase in neurogenesis in the Nestin-CreER T2 ;Jagged1 F/F mice is accompanied by a reduction in the Nestin + and Sox2 + cell populations

    Techniques Used: Mouse Assay

    7) Product Images from "bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling"

    Article Title: bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0307-1

    Proposed model for bFGF signaling in pluripotent (green) vs. differentiated (orange) hiPSCs (for details see discussion). bFGF, basic fibroblast growth factor; OCT4, octamer-binding transcription factor 4; SOX2, sex determining region Y-box 2; GFAP, glial fibrillary acidic protein; RAS, rat sarcoma; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositidedependent protein kinase; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3
    Figure Legend Snippet: Proposed model for bFGF signaling in pluripotent (green) vs. differentiated (orange) hiPSCs (for details see discussion). bFGF, basic fibroblast growth factor; OCT4, octamer-binding transcription factor 4; SOX2, sex determining region Y-box 2; GFAP, glial fibrillary acidic protein; RAS, rat sarcoma; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositidedependent protein kinase; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3

    Techniques Used: Binding Assay

    Long-term maintenance of undifferentiated hiPSCs. a Confocal imaging showed the expression of pluripotency markers (OCT4, SOX2, NANOG and SSEA4) and the absence of differentiation markers GFAP and α-SMA as ectodermal and mesodermal markers, respectively. Cell nuclei were stained with DAPI (blue). Scale bars, 10 μm. b Flow cytometry confirmed expression of OCT4, SSEA4 and SOX2 in hiPSCs with more than 98% of positive cells. c qPCR analysis for undifferentiated stem cell markers ( POU5F1 , SOX2 and NANOG ) and early commitment to differentiation markers ( BRACHYURY , PAX6 and AFP ). GAPDH was used as an internal control. d Immunoblot analysis showing the specificity of antibodies and expression of markers. HeLa cells were used as negative control, HFF and astrocytes were used as positive control for α-SMA and GFAP, respectively
    Figure Legend Snippet: Long-term maintenance of undifferentiated hiPSCs. a Confocal imaging showed the expression of pluripotency markers (OCT4, SOX2, NANOG and SSEA4) and the absence of differentiation markers GFAP and α-SMA as ectodermal and mesodermal markers, respectively. Cell nuclei were stained with DAPI (blue). Scale bars, 10 μm. b Flow cytometry confirmed expression of OCT4, SSEA4 and SOX2 in hiPSCs with more than 98% of positive cells. c qPCR analysis for undifferentiated stem cell markers ( POU5F1 , SOX2 and NANOG ) and early commitment to differentiation markers ( BRACHYURY , PAX6 and AFP ). GAPDH was used as an internal control. d Immunoblot analysis showing the specificity of antibodies and expression of markers. HeLa cells were used as negative control, HFF and astrocytes were used as positive control for α-SMA and GFAP, respectively

    Techniques Used: Imaging, Expressing, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    The critical role of bFGF for maintaining hiPSC pluripotency. a Phase contrast images of hiPSCs cultured under four different conditions, CM-100, CM-5, CM-0 and non-CM for 6 days. Undifferentiated hiPSCs formed compact colonies (CM-100), while without bFGF supplementation hiPSCs spread and flattened at day 6 (CM-0 and non-CM). Scale bar, 50 μm. b qPCR analysis showed the downregulation of pluripotency markers POU5F1 , SOX2 and NANOG in cells cultured in CM-0 and non-CM in comparison to control group (CM-100). All expression values were normalized to GAPDH . Results from three separate experiments, each carried out in triplicate, are shown as mean ± SD (ANOVA; * p
    Figure Legend Snippet: The critical role of bFGF for maintaining hiPSC pluripotency. a Phase contrast images of hiPSCs cultured under four different conditions, CM-100, CM-5, CM-0 and non-CM for 6 days. Undifferentiated hiPSCs formed compact colonies (CM-100), while without bFGF supplementation hiPSCs spread and flattened at day 6 (CM-0 and non-CM). Scale bar, 50 μm. b qPCR analysis showed the downregulation of pluripotency markers POU5F1 , SOX2 and NANOG in cells cultured in CM-0 and non-CM in comparison to control group (CM-100). All expression values were normalized to GAPDH . Results from three separate experiments, each carried out in triplicate, are shown as mean ± SD (ANOVA; * p

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    8) Product Images from "Function of Armcx3 and Armc10/SVH Genes in the Regulation of Progenitor Proliferation and Neural Differentiation in the Chicken Spinal Cord"

    Article Title: Function of Armcx3 and Armc10/SVH Genes in the Regulation of Progenitor Proliferation and Neural Differentiation in the Chicken Spinal Cord

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00047

    Armc10 overexpression reduces progenitors proliferation . Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 24hpe as described in Figure 1 . As is seen with Arcmx3, Armc10 protein is localized at the mitochondria of neural progenitors as indicated by its colocalization with the mitochondrial marker CoxIV (A) . (C) Schematic representation illustrating the distributions of the VZ and MZ at HH12+24hpe. Armc10 overexpression did not induce changes in the lateral distribution of electroporated neural progenitors (B,E) or in the percentage of GFP/Sox2 or GFP/HuCD positive cells (D,F,G) . However, it induced a reduction in the percentage of PH3/GFP and BrdU/GFP positive cells (H,I) . Data represent the mean ± s.e.m. ( * p
    Figure Legend Snippet: Armc10 overexpression reduces progenitors proliferation . Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 24hpe as described in Figure 1 . As is seen with Arcmx3, Armc10 protein is localized at the mitochondria of neural progenitors as indicated by its colocalization with the mitochondrial marker CoxIV (A) . (C) Schematic representation illustrating the distributions of the VZ and MZ at HH12+24hpe. Armc10 overexpression did not induce changes in the lateral distribution of electroporated neural progenitors (B,E) or in the percentage of GFP/Sox2 or GFP/HuCD positive cells (D,F,G) . However, it induced a reduction in the percentage of PH3/GFP and BrdU/GFP positive cells (H,I) . Data represent the mean ± s.e.m. ( * p

    Techniques Used: Over Expression, Marker

    Endogenous Armc10 silencing inhibits progenitor proliferation. (A–D) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with shControl and shArmc10 vectors and analyzed at 24hpe with the indicated immunostaining. No changes in the distribution of shArmc10 expressing cells are observed. (E,F) Armc10 silencing does not affect the percentage of HuC/D/GFP or Sox2/GFP-positive cells with respect to the control. (G,H) The percentage of GFP-positive electroporated cells positive for BrDU decreases in shArmc10 electroporated embryos; no changes are observed in the percentage of GFP-positive electroporated cells positive for PH3 with respect to the control. Data represent the mean ± s.e.m. ( * p
    Figure Legend Snippet: Endogenous Armc10 silencing inhibits progenitor proliferation. (A–D) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with shControl and shArmc10 vectors and analyzed at 24hpe with the indicated immunostaining. No changes in the distribution of shArmc10 expressing cells are observed. (E,F) Armc10 silencing does not affect the percentage of HuC/D/GFP or Sox2/GFP-positive cells with respect to the control. (G,H) The percentage of GFP-positive electroporated cells positive for BrDU decreases in shArmc10 electroporated embryos; no changes are observed in the percentage of GFP-positive electroporated cells positive for PH3 with respect to the control. Data represent the mean ± s.e.m. ( * p

    Techniques Used: Immunostaining, Expressing

    Endogenous Armc10 silencing inhibits progenitor proliferation but does not affect neural maturation. (A–F) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with shControl and shArmc10 plasmids and analyzed at 48hpe with the indicated immunostaining. No changes in the distribution of shArmc10 expressing cells (D) or in the percentage of HuC/D/GFP and Sox2/GFP-positive cells (F) are observed (anti-HuC/D, blue; anti-Sox2, red). (G,H) The HuC/D+ areas corresponding to the MZ (formed by the differentiating neurons) were defined using ImageJ processing (G) . The areas measured for the electroporated side (EP) were standardized to their contralateral controls (CNTRL) and are presented as ratios of the area of MZ (HuC/D + Area); the widths of the Tuj-1-marked region for the electroporated side were standardized to their contralateral controls and are presented as ratios of the size of MZ (Tuj + Size) (H) . (I) Histogram showing the percentage of electroporated cells (GFP + ) positive for Tuj-1. Data represent the mean ± s.e.m. ( * p
    Figure Legend Snippet: Endogenous Armc10 silencing inhibits progenitor proliferation but does not affect neural maturation. (A–F) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with shControl and shArmc10 plasmids and analyzed at 48hpe with the indicated immunostaining. No changes in the distribution of shArmc10 expressing cells (D) or in the percentage of HuC/D/GFP and Sox2/GFP-positive cells (F) are observed (anti-HuC/D, blue; anti-Sox2, red). (G,H) The HuC/D+ areas corresponding to the MZ (formed by the differentiating neurons) were defined using ImageJ processing (G) . The areas measured for the electroporated side (EP) were standardized to their contralateral controls (CNTRL) and are presented as ratios of the area of MZ (HuC/D + Area); the widths of the Tuj-1-marked region for the electroporated side were standardized to their contralateral controls and are presented as ratios of the size of MZ (Tuj + Size) (H) . (I) Histogram showing the percentage of electroporated cells (GFP + ) positive for Tuj-1. Data represent the mean ± s.e.m. ( * p

    Techniques Used: Immunostaining, Expressing

    Armcx3 overexpression promotes neural differentiation. (A–E) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with pCIG and pCIGArmcx3 vectors and analyzed at 48hpe with the indicated immunostaining. Armcx3 overexpressing cells show a lateral distribution from the VZ to the MZ of the neural tube. (F) Ectopic expression of Armcx3 leads to an increase in the percentage of HuC/D/GFP-positive cells and a decrease in Sox2/GFP-positive cells (anti-HuC/D, blue; anti-Sox2, red). (G) The HuC/D + areas corresponding to the MZ (formed by the differentiating neurons) were defined using ImageJ processing. (H) The areas measured for the electroporated side (EP) were standardized to their contralateral controls (CNTRL) and are presented as ratios of the area of MZ (HuC/D + Area); the widths of the Tuj-1-marked region for the electroporated side were standardized to their contralateral controls and are presented as ratios of the size of MZ (Tuj + Size). (I) Histogram showing the percentage of electroporated cells (GFP + ) positive for Tuj-1. Data represent the mean ± s.e.m. ( * p
    Figure Legend Snippet: Armcx3 overexpression promotes neural differentiation. (A–E) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with pCIG and pCIGArmcx3 vectors and analyzed at 48hpe with the indicated immunostaining. Armcx3 overexpressing cells show a lateral distribution from the VZ to the MZ of the neural tube. (F) Ectopic expression of Armcx3 leads to an increase in the percentage of HuC/D/GFP-positive cells and a decrease in Sox2/GFP-positive cells (anti-HuC/D, blue; anti-Sox2, red). (G) The HuC/D + areas corresponding to the MZ (formed by the differentiating neurons) were defined using ImageJ processing. (H) The areas measured for the electroporated side (EP) were standardized to their contralateral controls (CNTRL) and are presented as ratios of the area of MZ (HuC/D + Area); the widths of the Tuj-1-marked region for the electroporated side were standardized to their contralateral controls and are presented as ratios of the size of MZ (Tuj + Size). (I) Histogram showing the percentage of electroporated cells (GFP + ) positive for Tuj-1. Data represent the mean ± s.e.m. ( * p

    Techniques Used: Over Expression, Immunostaining, Expressing

    Armcx3 effects on progenitor proliferation depend on its mitochondrial localization. (A–D,F,H) Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 24hpe as described in Figure 1 . Overexpressed Arcmx3 is localized at the mitochondria of neural progenitors as indicated by its colocalization with the mitochondrial marker CoxIV. By contrast, a truncated form of Armcx3 which lacks its mitochondrial targeting sequence (Armcx3Δ1-12) displays a cytoplasmic non-mitochondrial localization (A) . Arrows in (A) label mitochondria co-localizing with Armcx3 (left panels) but not with the Armcx3Δ(1-12) construct lacking the mitochondrial targeting sequence (right panels). The overexpression of this truncated form neither induced a lateral distribution of progenitors (B,E) nor modified the percentage of Sox2/GFP-positive or HuCD/GFP-positive cells (D–G) . Consistently, the non-mitochondrial Armcx3 form did not induce changes in the percentage of GFP/PH3 or GFP/BrdU positive cells (I) . Data represent the mean ± s.e.m.
    Figure Legend Snippet: Armcx3 effects on progenitor proliferation depend on its mitochondrial localization. (A–D,F,H) Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 24hpe as described in Figure 1 . Overexpressed Arcmx3 is localized at the mitochondria of neural progenitors as indicated by its colocalization with the mitochondrial marker CoxIV. By contrast, a truncated form of Armcx3 which lacks its mitochondrial targeting sequence (Armcx3Δ1-12) displays a cytoplasmic non-mitochondrial localization (A) . Arrows in (A) label mitochondria co-localizing with Armcx3 (left panels) but not with the Armcx3Δ(1-12) construct lacking the mitochondrial targeting sequence (right panels). The overexpression of this truncated form neither induced a lateral distribution of progenitors (B,E) nor modified the percentage of Sox2/GFP-positive or HuCD/GFP-positive cells (D–G) . Consistently, the non-mitochondrial Armcx3 form did not induce changes in the percentage of GFP/PH3 or GFP/BrdU positive cells (I) . Data represent the mean ± s.e.m.

    Techniques Used: Marker, Sequencing, Construct, Over Expression, Modification

    Armcx3 effects on neural differentiation depend on its mitochondrial localization. (A,C,E) Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 48hpe as described in Figure 2 . In contrast to the full length Armcx3 (see Figure 2 ), the overexpression of a non-mitochondrial truncated version of Arcmx3 (Armcx3Δ1-12) did not induce apparent changes in the lateral distribution of progenitors (A–D) or modify the percentage of Sox2/GFP or HuCD/GFP positive cells. (F) Similarly, Armcx3Δ1-12 overexpression has no impact on the HuCD + area (G,H) , the width of Tuj-1 positive region (G,H) , or the percentage of electroporated cells positive for Tuj-1 (I) . Data represent the mean ± s.e.m.
    Figure Legend Snippet: Armcx3 effects on neural differentiation depend on its mitochondrial localization. (A,C,E) Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 48hpe as described in Figure 2 . In contrast to the full length Armcx3 (see Figure 2 ), the overexpression of a non-mitochondrial truncated version of Arcmx3 (Armcx3Δ1-12) did not induce apparent changes in the lateral distribution of progenitors (A–D) or modify the percentage of Sox2/GFP or HuCD/GFP positive cells. (F) Similarly, Armcx3Δ1-12 overexpression has no impact on the HuCD + area (G,H) , the width of Tuj-1 positive region (G,H) , or the percentage of electroporated cells positive for Tuj-1 (I) . Data represent the mean ± s.e.m.

    Techniques Used: Over Expression

    Armc10 overexpression does not promote neural differentiation . Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 48hpe as described in Figure 2 . The overexpression of Armc10 did not induce changes in the lateral distribution of electroporated progenitors (A) or modify the percentage of Sox2/GFP or HuCD/GFP positive cells (C,D) . By contrast, Armc10 overexpression reduced the HuCD area and the width of Tuj-1 positive region (G,H) . The percentage of electroporated cells positive for Tuj-1 was not different in embryos where Armc10 was overexpressed with respect to controls (pCIG) (I) . Data represent the mean ± s.e.m. ( * p
    Figure Legend Snippet: Armc10 overexpression does not promote neural differentiation . Representative transverse sections of chick neural tubes from embryos electroporated at HH stage 12 with the indicated plasmids and processed and analyzed 48hpe as described in Figure 2 . The overexpression of Armc10 did not induce changes in the lateral distribution of electroporated progenitors (A) or modify the percentage of Sox2/GFP or HuCD/GFP positive cells (C,D) . By contrast, Armc10 overexpression reduced the HuCD area and the width of Tuj-1 positive region (G,H) . The percentage of electroporated cells positive for Tuj-1 was not different in embryos where Armc10 was overexpressed with respect to controls (pCIG) (I) . Data represent the mean ± s.e.m. ( * p

    Techniques Used: Over Expression

    Armcx3 overexpression reduces progenitor proliferation. (A–D) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with pCIG and pCIGArmcx3 vectors and analyzed at 24hpe with the indicated immunostaining. GFP, Sox2 (red) and HuC/D (blue) stain, respectively, the electroporated cells, the neural progenitors, and the differentiating neurons; Armcx3 overexpressing cells show a lateral distribution from the lumen to the MZ of the neural tube. (E,F) Ectopic expression of Armcx3 leads to a dramatic increase in the percentage of HuC/D/GFP-positive cells, concomitant to a decrease in Sox2/GFP-positive cells. (G,H) The percentage of GFP-positive electroporated cells positive for PH3 or BrDU decreases in pCIGArmcx3 electroporated embryos. Data represent the mean ± s.e.m. ( * p
    Figure Legend Snippet: Armcx3 overexpression reduces progenitor proliferation. (A–D) Representative transverse sections of neural tubes from embryos electroporated at HH stage 12 with pCIG and pCIGArmcx3 vectors and analyzed at 24hpe with the indicated immunostaining. GFP, Sox2 (red) and HuC/D (blue) stain, respectively, the electroporated cells, the neural progenitors, and the differentiating neurons; Armcx3 overexpressing cells show a lateral distribution from the lumen to the MZ of the neural tube. (E,F) Ectopic expression of Armcx3 leads to a dramatic increase in the percentage of HuC/D/GFP-positive cells, concomitant to a decrease in Sox2/GFP-positive cells. (G,H) The percentage of GFP-positive electroporated cells positive for PH3 or BrDU decreases in pCIGArmcx3 electroporated embryos. Data represent the mean ± s.e.m. ( * p

    Techniques Used: Over Expression, Immunostaining, Staining, Expressing

    9) Product Images from "Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis"

    Article Title: Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1000460

    Adult NSCs in the SGZ do not self-maintain in the absence of intermediate progenitors. Nestin (B, C), Sox2 (E, F), and Id1/PECAM (H, I) immunostaining of the subgranular zone (SGZ) of 4-mo-old controls (B, E, H) and Nestin-CreER T2 ;Prox1 F/F (C, F, I) mice treated with TM from P0 to P15. Graphs compare the number of Nestin + (A), Sox2 + (D), and Id1 + PECAM − (G) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice treated with TM from P0 to P15. At P20, the number of Nestin + , Sox2 + , or Id1 + PECAM − NSCs is similar in controls and Nestin-CreER T2 ;Prox1 F/F brains (A, D, G). However, after P20, the number of Nestin + (A–C), Sox2 + (D–F), or Id1 + PECAM − (G–I) cells is reduced in Nestin-CreER T2 ;Prox1 F/F mice. Graphs compare the number of Nestin + (J), Sox2 + (M), and Id1 + PECAM − (P) cells in 12-wk-old and 16-wk-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice treated with TM for 4 wk starting at 8 wk of age. The number of Nestin + , Sox2 + , and Id1 + PECAM − adult NSCs was increased in 12-wk-old conditional mutant brains (J, M, P). At 16 wk, the number of Nestin + (J, L), Sox2 + (M, O), and Id1 + PECAM - (P, R) cells is reduced in the SGZ of conditional-mutant mice. The percentage of Nestin + /b-Gal + and Sox2 + /b-Gal + cells is similar in 12-wk-old (S) and 16-wk-old (T) control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) brains that carry the ROSA allele. However, the percentage of Tbr2 + /b-Gal + and Dcx + /b-Gal + cells is reduced at both stages in the Nestin-CreER T2 ;Prox1 F/F ; ROSA SGZ (S, T). Data represent the mean number of positive cells per DG section ± SD. N = 4 brains. Paired t test. * p
    Figure Legend Snippet: Adult NSCs in the SGZ do not self-maintain in the absence of intermediate progenitors. Nestin (B, C), Sox2 (E, F), and Id1/PECAM (H, I) immunostaining of the subgranular zone (SGZ) of 4-mo-old controls (B, E, H) and Nestin-CreER T2 ;Prox1 F/F (C, F, I) mice treated with TM from P0 to P15. Graphs compare the number of Nestin + (A), Sox2 + (D), and Id1 + PECAM − (G) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice treated with TM from P0 to P15. At P20, the number of Nestin + , Sox2 + , or Id1 + PECAM − NSCs is similar in controls and Nestin-CreER T2 ;Prox1 F/F brains (A, D, G). However, after P20, the number of Nestin + (A–C), Sox2 + (D–F), or Id1 + PECAM − (G–I) cells is reduced in Nestin-CreER T2 ;Prox1 F/F mice. Graphs compare the number of Nestin + (J), Sox2 + (M), and Id1 + PECAM − (P) cells in 12-wk-old and 16-wk-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice treated with TM for 4 wk starting at 8 wk of age. The number of Nestin + , Sox2 + , and Id1 + PECAM − adult NSCs was increased in 12-wk-old conditional mutant brains (J, M, P). At 16 wk, the number of Nestin + (J, L), Sox2 + (M, O), and Id1 + PECAM - (P, R) cells is reduced in the SGZ of conditional-mutant mice. The percentage of Nestin + /b-Gal + and Sox2 + /b-Gal + cells is similar in 12-wk-old (S) and 16-wk-old (T) control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) brains that carry the ROSA allele. However, the percentage of Tbr2 + /b-Gal + and Dcx + /b-Gal + cells is reduced at both stages in the Nestin-CreER T2 ;Prox1 F/F ; ROSA SGZ (S, T). Data represent the mean number of positive cells per DG section ± SD. N = 4 brains. Paired t test. * p

    Techniques Used: Immunostaining, Mouse Assay, Mutagenesis

    Prox1 ectopic-expression promotes NSC premature differentiation. Anti-C-Prox1 staining (A, control) shows a reduced dentate gyrus (DG) in P30 Nestin-Cre;JoJo(Prox1) mice (B, V). There is no difference in the number of Prox1 + cells in the DG region of control (C, E) and Nestin-Cre;JoJo(Prox1) (D, E) at E16.5. There are no differences either in number of Sox2 + (F) or Ki67 + (G) cells in the dentate neuroepithelium (DNE) and the DG at E16.5. At P8, there is an increase in the number of Dcx + (H, I) and NeuN + (J, L) cells in TM-treated Nestin-CreER T2 ;Prox1 F/F brains. However, the number of Nestin + (M, N) cells is reduced at this stage. (O) Cell fate analysis of the Prox1 ectopic-expressing cells, showing that they predominantly differentiate towards a neuronal lineage. The number of Nestin + adult NSCs (P, Q, T) and Dcx + intermediate progenitors (R, S, U) is reduced in the SGZ of P30 Nestin-Cre;JoJo-Prox1 animals. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. *** p
    Figure Legend Snippet: Prox1 ectopic-expression promotes NSC premature differentiation. Anti-C-Prox1 staining (A, control) shows a reduced dentate gyrus (DG) in P30 Nestin-Cre;JoJo(Prox1) mice (B, V). There is no difference in the number of Prox1 + cells in the DG region of control (C, E) and Nestin-Cre;JoJo(Prox1) (D, E) at E16.5. There are no differences either in number of Sox2 + (F) or Ki67 + (G) cells in the dentate neuroepithelium (DNE) and the DG at E16.5. At P8, there is an increase in the number of Dcx + (H, I) and NeuN + (J, L) cells in TM-treated Nestin-CreER T2 ;Prox1 F/F brains. However, the number of Nestin + (M, N) cells is reduced at this stage. (O) Cell fate analysis of the Prox1 ectopic-expressing cells, showing that they predominantly differentiate towards a neuronal lineage. The number of Nestin + adult NSCs (P, Q, T) and Dcx + intermediate progenitors (R, S, U) is reduced in the SGZ of P30 Nestin-Cre;JoJo-Prox1 animals. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. *** p

    Techniques Used: Expressing, Staining, Mouse Assay

    Prox1 is required in the subgranular zone during adult neurogenesis. During adult neurogenesis, Prox1 is not expressed in Nestin + (A), Sox2 + (B), or Id1 + (C) adult neural stem cells or in Acsl + Type-IIa intermediate progenitors (D). Instead, Prox1 is expressed in Tbr2 + (E) and Dcx + Type-IIb intermediate progenitors (F), Type-III intermediate progenitors (G), and Calretinin + immature neurons. Graphs compare the number of Tbr2 + (K) Dcx + (N) and Calretinin + (Q) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice. A reduced number of Tbr2 + (J, K), Dcx + (M, N), and Calretinin + (P, Q) cells was observed in the SGZ of 4-mo-old Nestin-CreER T2 ;Prox1 F/F mice. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. * p
    Figure Legend Snippet: Prox1 is required in the subgranular zone during adult neurogenesis. During adult neurogenesis, Prox1 is not expressed in Nestin + (A), Sox2 + (B), or Id1 + (C) adult neural stem cells or in Acsl + Type-IIa intermediate progenitors (D). Instead, Prox1 is expressed in Tbr2 + (E) and Dcx + Type-IIb intermediate progenitors (F), Type-III intermediate progenitors (G), and Calretinin + immature neurons. Graphs compare the number of Tbr2 + (K) Dcx + (N) and Calretinin + (Q) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice. A reduced number of Tbr2 + (J, K), Dcx + (M, N), and Calretinin + (P, Q) cells was observed in the SGZ of 4-mo-old Nestin-CreER T2 ;Prox1 F/F mice. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. * p

    Techniques Used: Mouse Assay

    Post-natal lack of Prox1 affects dentate gyrus formation. Hematoxylin and eosin staining (A, B) of coronal sections of the hippocampus shows a reduced dentate gyrus (DG) in 2-mo-old Nestin-CreER T2 ;Prox1 F/F mice treated with TM from P0 to P15 (B). There are no changes in the neural stem cell population at P10, as indicated by Nestin (C–E), Sox2 (F–H), and Id1 (I–K) IHC in the Nestin-CreER T2 ;Prox1 F/F DG. BrdU staining after a 1-h pulse shows a similar level of proliferation in the DG of wild-type (L, N) and Nestin CreER T2 ;Prox1 F/F brains (M, N). The number of Tbr2 + intermediate progenitors (O–Q) and Dcx + (R, S) cells is reduced in the Nestin-CreER T2 ;Prox1 F/F DG at P10. We also observed an increase in the number of TUNEL + cells (T–V) in the DG area of the Nestin-CreER T2 ;Prox1 F/F brains at P10. Data represent the mean number of positive cells per DG section ± SD ( N = 3 mice). Blue bars are controls. Red bars are TM-treated Nestin CreER T2 ;Prox1 F/F brains. Paired t test. * p
    Figure Legend Snippet: Post-natal lack of Prox1 affects dentate gyrus formation. Hematoxylin and eosin staining (A, B) of coronal sections of the hippocampus shows a reduced dentate gyrus (DG) in 2-mo-old Nestin-CreER T2 ;Prox1 F/F mice treated with TM from P0 to P15 (B). There are no changes in the neural stem cell population at P10, as indicated by Nestin (C–E), Sox2 (F–H), and Id1 (I–K) IHC in the Nestin-CreER T2 ;Prox1 F/F DG. BrdU staining after a 1-h pulse shows a similar level of proliferation in the DG of wild-type (L, N) and Nestin CreER T2 ;Prox1 F/F brains (M, N). The number of Tbr2 + intermediate progenitors (O–Q) and Dcx + (R, S) cells is reduced in the Nestin-CreER T2 ;Prox1 F/F DG at P10. We also observed an increase in the number of TUNEL + cells (T–V) in the DG area of the Nestin-CreER T2 ;Prox1 F/F brains at P10. Data represent the mean number of positive cells per DG section ± SD ( N = 3 mice). Blue bars are controls. Red bars are TM-treated Nestin CreER T2 ;Prox1 F/F brains. Paired t test. * p

    Techniques Used: Staining, Mouse Assay, Immunohistochemistry, BrdU Staining, TUNEL Assay

    10) Product Images from "Regulation of ES Cell Self Renewal and Pluripotency by Foxd3"

    Article Title: Regulation of ES Cell Self Renewal and Pluripotency by Foxd3

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1634/stemcells.2008-0269

    Foxd3 represses ES cell differentiation towards multiple lineages (A): qRT-PCR analysis of transcript levels of primitive endoderm markers Foxa2 , AFP and Sox17 in ES cells cultured with or without TM for 3 days. Bars represent transcript levels for each gene in 2μM TM treated cells, and horizontal lines indicate the mRNA levels in untreated ES cells arbitrarily designated as 1. (B): qRT-PCR analysis of mRNA levels of trophectoderm markers Cdx2 , Fgfr2 and Csh1/PL1 . (C): qRT-PCR analysis of mRNA levels of epiblast marker Fgf5 , mesendoderm markers T and Gsc . (D): qRT-PCR analysis of neurectoderm markers Sox1 Nestin , and Pax6 . (E): Immunocytochemistry of Foxa2 (red) and either Sox2 or Oct4 (green) in Foxd3 fl/fl ; Cre-ER ES cells treated with 2μM TM for 3 days. Arrowheads: Sox2/Foxa2 double-positive cells. Scale bar: 50μm. (F): Percent of Oct4- or Foxa2-positive cells in control and 2μM TM-treated cultures. Data were collected from three independent experiments, and > 2000 cells were counted for each treatment group. (G): TUNEL labeling (green) and immunocytochemistry for either Oct4 or Foxa2 (red) in Foxd3 fl/fl ; Cre-ER ES cells treated with 2μM TM for 3 days. Arrowheads: TUNEL/Foxa2 double-positive cells that are also DAPI-positive. Arrows: TUNEL/Foxa2 double-positive cells that are DAPI-negative indicating they have lost their nuclear integrity. Scale bar: 25 μm. (H): Percent of TUNEL-positive cells in Oct4-positive or Foxa2-positive populations in control and mutant cultures. More than 2000 cells were counted for each treatment group. Data shown are Mean±SEM (*** p
    Figure Legend Snippet: Foxd3 represses ES cell differentiation towards multiple lineages (A): qRT-PCR analysis of transcript levels of primitive endoderm markers Foxa2 , AFP and Sox17 in ES cells cultured with or without TM for 3 days. Bars represent transcript levels for each gene in 2μM TM treated cells, and horizontal lines indicate the mRNA levels in untreated ES cells arbitrarily designated as 1. (B): qRT-PCR analysis of mRNA levels of trophectoderm markers Cdx2 , Fgfr2 and Csh1/PL1 . (C): qRT-PCR analysis of mRNA levels of epiblast marker Fgf5 , mesendoderm markers T and Gsc . (D): qRT-PCR analysis of neurectoderm markers Sox1 Nestin , and Pax6 . (E): Immunocytochemistry of Foxa2 (red) and either Sox2 or Oct4 (green) in Foxd3 fl/fl ; Cre-ER ES cells treated with 2μM TM for 3 days. Arrowheads: Sox2/Foxa2 double-positive cells. Scale bar: 50μm. (F): Percent of Oct4- or Foxa2-positive cells in control and 2μM TM-treated cultures. Data were collected from three independent experiments, and > 2000 cells were counted for each treatment group. (G): TUNEL labeling (green) and immunocytochemistry for either Oct4 or Foxa2 (red) in Foxd3 fl/fl ; Cre-ER ES cells treated with 2μM TM for 3 days. Arrowheads: TUNEL/Foxa2 double-positive cells that are also DAPI-positive. Arrows: TUNEL/Foxa2 double-positive cells that are DAPI-negative indicating they have lost their nuclear integrity. Scale bar: 25 μm. (H): Percent of TUNEL-positive cells in Oct4-positive or Foxa2-positive populations in control and mutant cultures. More than 2000 cells were counted for each treatment group. Data shown are Mean±SEM (*** p

    Techniques Used: Cell Differentiation, Quantitative RT-PCR, Cell Culture, Marker, Immunocytochemistry, TUNEL Assay, Labeling, Mutagenesis

    Foxd3 is not required for the expression of stem cell markers (A): Immunocytochemistry shows nuclear staining of Oct4, Sox2 and Nanog (red) in Foxd3 fl/fl ; Cre-ER ES cells with or without 2μM TM treatment for 3 days. ES cells were counter-stained with DAPI (blue). Oct4 (red) and Foxd3 (green) double staining (leftmost panel) reveals that most Foxd3 -deficient ES cells are Oct4-positive in mutant cultures and very little Foxd3 protein is observed in treated cells. Scale bar: 50μm. (B): qRT-PCR analysis of stem cell marker gene expression in 2μM TM (red line) or no TM (blue line) treated cells for 1-3 days. mRNA levels of each gene are compared between control and TM-treated cells by Student's t-test (***: p
    Figure Legend Snippet: Foxd3 is not required for the expression of stem cell markers (A): Immunocytochemistry shows nuclear staining of Oct4, Sox2 and Nanog (red) in Foxd3 fl/fl ; Cre-ER ES cells with or without 2μM TM treatment for 3 days. ES cells were counter-stained with DAPI (blue). Oct4 (red) and Foxd3 (green) double staining (leftmost panel) reveals that most Foxd3 -deficient ES cells are Oct4-positive in mutant cultures and very little Foxd3 protein is observed in treated cells. Scale bar: 50μm. (B): qRT-PCR analysis of stem cell marker gene expression in 2μM TM (red line) or no TM (blue line) treated cells for 1-3 days. mRNA levels of each gene are compared between control and TM-treated cells by Student's t-test (***: p

    Techniques Used: Expressing, Immunocytochemistry, Staining, Double Staining, Mutagenesis, Quantitative RT-PCR, Marker

    11) Product Images from "Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis"

    Article Title: Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1000460

    Adult NSCs in the SGZ do not self-maintain in the absence of intermediate progenitors. Nestin (B, C), Sox2 (E, F), and Id1/PECAM (H, I) immunostaining of the subgranular zone (SGZ) of 4-mo-old controls (B, E, H) and Nestin-CreER T2 ;Prox1 F/F (C, F, I) mice treated with TM from P0 to P15. Graphs compare the number of Nestin + (A), Sox2 + (D), and Id1 + PECAM − (G) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice treated with TM from P0 to P15. At P20, the number of Nestin + , Sox2 + , or Id1 + PECAM − NSCs is similar in controls and Nestin-CreER T2 ;Prox1 F/F brains (A, D, G). However, after P20, the number of Nestin + (A–C), Sox2 + (D–F), or Id1 + PECAM − (G–I) cells is reduced in Nestin-CreER T2 ;Prox1 F/F mice. Graphs compare the number of Nestin + (J), Sox2 + (M), and Id1 + PECAM − (P) cells in 12-wk-old and 16-wk-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice treated with TM for 4 wk starting at 8 wk of age. The number of Nestin + , Sox2 + , and Id1 + PECAM − adult NSCs was increased in 12-wk-old conditional mutant brains (J, M, P). At 16 wk, the number of Nestin + (J, L), Sox2 + (M, O), and Id1 + PECAM - (P, R) cells is reduced in the SGZ of conditional-mutant mice. The percentage of Nestin + /b-Gal + and Sox2 + /b-Gal + cells is similar in 12-wk-old (S) and 16-wk-old (T) control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) brains that carry the ROSA allele. However, the percentage of Tbr2 + /b-Gal + and Dcx + /b-Gal + cells is reduced at both stages in the Nestin-CreER T2 ;Prox1 F/F ; ROSA SGZ (S, T). Data represent the mean number of positive cells per DG section ± SD. N = 4 brains. Paired t test. * p
    Figure Legend Snippet: Adult NSCs in the SGZ do not self-maintain in the absence of intermediate progenitors. Nestin (B, C), Sox2 (E, F), and Id1/PECAM (H, I) immunostaining of the subgranular zone (SGZ) of 4-mo-old controls (B, E, H) and Nestin-CreER T2 ;Prox1 F/F (C, F, I) mice treated with TM from P0 to P15. Graphs compare the number of Nestin + (A), Sox2 + (D), and Id1 + PECAM − (G) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice treated with TM from P0 to P15. At P20, the number of Nestin + , Sox2 + , or Id1 + PECAM − NSCs is similar in controls and Nestin-CreER T2 ;Prox1 F/F brains (A, D, G). However, after P20, the number of Nestin + (A–C), Sox2 + (D–F), or Id1 + PECAM − (G–I) cells is reduced in Nestin-CreER T2 ;Prox1 F/F mice. Graphs compare the number of Nestin + (J), Sox2 + (M), and Id1 + PECAM − (P) cells in 12-wk-old and 16-wk-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice treated with TM for 4 wk starting at 8 wk of age. The number of Nestin + , Sox2 + , and Id1 + PECAM − adult NSCs was increased in 12-wk-old conditional mutant brains (J, M, P). At 16 wk, the number of Nestin + (J, L), Sox2 + (M, O), and Id1 + PECAM - (P, R) cells is reduced in the SGZ of conditional-mutant mice. The percentage of Nestin + /b-Gal + and Sox2 + /b-Gal + cells is similar in 12-wk-old (S) and 16-wk-old (T) control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) brains that carry the ROSA allele. However, the percentage of Tbr2 + /b-Gal + and Dcx + /b-Gal + cells is reduced at both stages in the Nestin-CreER T2 ;Prox1 F/F ; ROSA SGZ (S, T). Data represent the mean number of positive cells per DG section ± SD. N = 4 brains. Paired t test. * p

    Techniques Used: Immunostaining, Mouse Assay, Mutagenesis

    Prox1 ectopic-expression promotes NSC premature differentiation. Anti-C-Prox1 staining (A, control) shows a reduced dentate gyrus (DG) in P30 Nestin-Cre;JoJo(Prox1) mice (B, V). There is no difference in the number of Prox1 + cells in the DG region of control (C, E) and Nestin-Cre;JoJo(Prox1) (D, E) at E16.5. There are no differences either in number of Sox2 + (F) or Ki67 + (G) cells in the dentate neuroepithelium (DNE) and the DG at E16.5. At P8, there is an increase in the number of Dcx + (H, I) and NeuN + (J, L) cells in TM-treated Nestin-CreER T2 ;Prox1 F/F brains. However, the number of Nestin + (M, N) cells is reduced at this stage. (O) Cell fate analysis of the Prox1 ectopic-expressing cells, showing that they predominantly differentiate towards a neuronal lineage. The number of Nestin + adult NSCs (P, Q, T) and Dcx + intermediate progenitors (R, S, U) is reduced in the SGZ of P30 Nestin-Cre;JoJo-Prox1 animals. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. *** p
    Figure Legend Snippet: Prox1 ectopic-expression promotes NSC premature differentiation. Anti-C-Prox1 staining (A, control) shows a reduced dentate gyrus (DG) in P30 Nestin-Cre;JoJo(Prox1) mice (B, V). There is no difference in the number of Prox1 + cells in the DG region of control (C, E) and Nestin-Cre;JoJo(Prox1) (D, E) at E16.5. There are no differences either in number of Sox2 + (F) or Ki67 + (G) cells in the dentate neuroepithelium (DNE) and the DG at E16.5. At P8, there is an increase in the number of Dcx + (H, I) and NeuN + (J, L) cells in TM-treated Nestin-CreER T2 ;Prox1 F/F brains. However, the number of Nestin + (M, N) cells is reduced at this stage. (O) Cell fate analysis of the Prox1 ectopic-expressing cells, showing that they predominantly differentiate towards a neuronal lineage. The number of Nestin + adult NSCs (P, Q, T) and Dcx + intermediate progenitors (R, S, U) is reduced in the SGZ of P30 Nestin-Cre;JoJo-Prox1 animals. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. *** p

    Techniques Used: Expressing, Staining, Mouse Assay

    Prox1 is required in the subgranular zone during adult neurogenesis. During adult neurogenesis, Prox1 is not expressed in Nestin + (A), Sox2 + (B), or Id1 + (C) adult neural stem cells or in Acsl + Type-IIa intermediate progenitors (D). Instead, Prox1 is expressed in Tbr2 + (E) and Dcx + Type-IIb intermediate progenitors (F), Type-III intermediate progenitors (G), and Calretinin + immature neurons. Graphs compare the number of Tbr2 + (K) Dcx + (N) and Calretinin + (Q) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice. A reduced number of Tbr2 + (J, K), Dcx + (M, N), and Calretinin + (P, Q) cells was observed in the SGZ of 4-mo-old Nestin-CreER T2 ;Prox1 F/F mice. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. * p
    Figure Legend Snippet: Prox1 is required in the subgranular zone during adult neurogenesis. During adult neurogenesis, Prox1 is not expressed in Nestin + (A), Sox2 + (B), or Id1 + (C) adult neural stem cells or in Acsl + Type-IIa intermediate progenitors (D). Instead, Prox1 is expressed in Tbr2 + (E) and Dcx + Type-IIb intermediate progenitors (F), Type-III intermediate progenitors (G), and Calretinin + immature neurons. Graphs compare the number of Tbr2 + (K) Dcx + (N) and Calretinin + (Q) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER T2 ;Prox1 F/F (red) mice. A reduced number of Tbr2 + (J, K), Dcx + (M, N), and Calretinin + (P, Q) cells was observed in the SGZ of 4-mo-old Nestin-CreER T2 ;Prox1 F/F mice. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. * p

    Techniques Used: Mouse Assay

    Post-natal lack of Prox1 affects dentate gyrus formation. Hematoxylin and eosin staining (A, B) of coronal sections of the hippocampus shows a reduced dentate gyrus (DG) in 2-mo-old Nestin-CreER T2 ;Prox1 F/F mice treated with TM from P0 to P15 (B). There are no changes in the neural stem cell population at P10, as indicated by Nestin (C–E), Sox2 (F–H), and Id1 (I–K) IHC in the Nestin-CreER T2 ;Prox1 F/F DG. BrdU staining after a 1-h pulse shows a similar level of proliferation in the DG of wild-type (L, N) and Nestin CreER T2 ;Prox1 F/F brains (M, N). The number of Tbr2 + intermediate progenitors (O–Q) and Dcx + (R, S) cells is reduced in the Nestin-CreER T2 ;Prox1 F/F DG at P10. We also observed an increase in the number of TUNEL + cells (T–V) in the DG area of the Nestin-CreER T2 ;Prox1 F/F brains at P10. Data represent the mean number of positive cells per DG section ± SD ( N = 3 mice). Blue bars are controls. Red bars are TM-treated Nestin CreER T2 ;Prox1 F/F brains. Paired t test. * p
    Figure Legend Snippet: Post-natal lack of Prox1 affects dentate gyrus formation. Hematoxylin and eosin staining (A, B) of coronal sections of the hippocampus shows a reduced dentate gyrus (DG) in 2-mo-old Nestin-CreER T2 ;Prox1 F/F mice treated with TM from P0 to P15 (B). There are no changes in the neural stem cell population at P10, as indicated by Nestin (C–E), Sox2 (F–H), and Id1 (I–K) IHC in the Nestin-CreER T2 ;Prox1 F/F DG. BrdU staining after a 1-h pulse shows a similar level of proliferation in the DG of wild-type (L, N) and Nestin CreER T2 ;Prox1 F/F brains (M, N). The number of Tbr2 + intermediate progenitors (O–Q) and Dcx + (R, S) cells is reduced in the Nestin-CreER T2 ;Prox1 F/F DG at P10. We also observed an increase in the number of TUNEL + cells (T–V) in the DG area of the Nestin-CreER T2 ;Prox1 F/F brains at P10. Data represent the mean number of positive cells per DG section ± SD ( N = 3 mice). Blue bars are controls. Red bars are TM-treated Nestin CreER T2 ;Prox1 F/F brains. Paired t test. * p

    Techniques Used: Staining, Mouse Assay, Immunohistochemistry, BrdU Staining, TUNEL Assay

    12) Product Images from "Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium"

    Article Title: Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054552

    Efficient generation of polarized neural progenitors from hESCs in a Matrigel-based 3D neuroepithelial cyst model. (A) Schematic of the experiment. (B) hESC clumps found at Day 0 formed neural tube-like cysts with a single lumen by Day 5. (C) By Day 1, hESC-derived cysts were positive for SOX2 and NESTIN. The apical localization of CD133 indicates apicobasal polarity is firmly established. (D) Immunostaining of PAX6 and ZO-1 during cyst growth in Matrigel. PAX6 was strongly expressed in Day 5 cysts that display clear properties of a pseudostratified epithelium. (E) M-phase cells stained with Phospho-Histone H3 (PH3) antibody only localized at the apical side of the cysts and S-phase cells labeled with EdU at the basolateral side, indicating that luminal mitosis occurred within the cysts. Nuclei were counterstained with Hoechst. Scale bar, 50 µm.
    Figure Legend Snippet: Efficient generation of polarized neural progenitors from hESCs in a Matrigel-based 3D neuroepithelial cyst model. (A) Schematic of the experiment. (B) hESC clumps found at Day 0 formed neural tube-like cysts with a single lumen by Day 5. (C) By Day 1, hESC-derived cysts were positive for SOX2 and NESTIN. The apical localization of CD133 indicates apicobasal polarity is firmly established. (D) Immunostaining of PAX6 and ZO-1 during cyst growth in Matrigel. PAX6 was strongly expressed in Day 5 cysts that display clear properties of a pseudostratified epithelium. (E) M-phase cells stained with Phospho-Histone H3 (PH3) antibody only localized at the apical side of the cysts and S-phase cells labeled with EdU at the basolateral side, indicating that luminal mitosis occurred within the cysts. Nuclei were counterstained with Hoechst. Scale bar, 50 µm.

    Techniques Used: Derivative Assay, Immunostaining, Staining, Labeling

    Related Articles

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    Immunocytochemistry:

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    Expressing:

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    Blocking Assay:

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    Article Snippet: .. Slides were subsequently incubated over night at 4°C with the following primary antibodies diluted in blocking buffer: Mouse antinestin (clone 401; BD Bioscience, cat# 556309; 1:1,000) and rabbit anti-Sox2 (Thermo Scientific, cat# PA1-16968; 1:250) were used to stain neural stem/progenitors; rabbit anti-Ki67 (Novocastra, cat# NSL-Ki67p; 1:8,000) to stain proliferating cells; rabbit anti-glial fibrillary acidic protein (GFAP; Chemicon, cat# AB5804; 1:1,000 or Sigma-Aldrich, cat# G9269; 1:1,000) or mouse anti-GFAP (clone GA-5; Cell Signaling, cat# 3670; 1:500) to stain astrocytes; mouse anti-oligodendrocytes (RIP, clone NSC-1; Chemicon, cat# MAB1580; 1:20,000) and mouse anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (2′,3′-cyclic nucleotide 3′-phospho-diesterase [CNPase], clone 11-5B; Sigma-Aldrich, cat# C5922; 1:100) to stain oligodendrocytes; mouse anti-β-III tubulin (TUJ1; Covance, Cat# MMS-435P; 1:1,000) or rabbit anti-β-III tubulin (Abcam, cat# AB18207; 1:2,000) to stain neurons; rabbit anti-Jagged 1 (Abcam, cat#Ab7771; 1:200) and goat anti-Notch 1 (R & D Systems, cat# AF1057; 1:100) to stain Notch signaling proteins. .. After PBS washes, coverslips were incubated with flourescent dye-conjugated secondary antibodies for 1 h at room temperature.

    Incubation:

    Article Title: miR-590-3p and Its Downstream Target Genes in HCC Cell Lines
    Article Snippet: .. For ICC, blocking was performed again and the cells were incubated with anti-SOX2 (1 : 250, PA1-16968, Thermo Scientific, USA), followed by the secondary antibody, Goat anti-rabbit IgG (H+L) DyLight 488 Conjugated (1 : 250, 35552, Thermo Scientific, USA). .. The nuclei were stained with DAPI (1 : 1000 in PBS, KPL, USA).

    Article Title: Human Stem/Progenitor Cells from Bone Marrow Enhance Glial Differentiation of Rat Neural Stem Cells: A Role for Transforming Growth Factor ? and Notch Signaling
    Article Snippet: .. Slides were subsequently incubated over night at 4°C with the following primary antibodies diluted in blocking buffer: Mouse antinestin (clone 401; BD Bioscience, cat# 556309; 1:1,000) and rabbit anti-Sox2 (Thermo Scientific, cat# PA1-16968; 1:250) were used to stain neural stem/progenitors; rabbit anti-Ki67 (Novocastra, cat# NSL-Ki67p; 1:8,000) to stain proliferating cells; rabbit anti-glial fibrillary acidic protein (GFAP; Chemicon, cat# AB5804; 1:1,000 or Sigma-Aldrich, cat# G9269; 1:1,000) or mouse anti-GFAP (clone GA-5; Cell Signaling, cat# 3670; 1:500) to stain astrocytes; mouse anti-oligodendrocytes (RIP, clone NSC-1; Chemicon, cat# MAB1580; 1:20,000) and mouse anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (2′,3′-cyclic nucleotide 3′-phospho-diesterase [CNPase], clone 11-5B; Sigma-Aldrich, cat# C5922; 1:100) to stain oligodendrocytes; mouse anti-β-III tubulin (TUJ1; Covance, Cat# MMS-435P; 1:1,000) or rabbit anti-β-III tubulin (Abcam, cat# AB18207; 1:2,000) to stain neurons; rabbit anti-Jagged 1 (Abcam, cat#Ab7771; 1:200) and goat anti-Notch 1 (R & D Systems, cat# AF1057; 1:100) to stain Notch signaling proteins. .. After PBS washes, coverslips were incubated with flourescent dye-conjugated secondary antibodies for 1 h at room temperature.

    Article Title: Altered transcriptional regulatory proteins in glioblastoma and YBX1 as a potential regulator of tumor invasion
    Article Snippet: .. Sections were then incubated for 1.5 h at room temperature with primary antibodies - HMGB2 (Abcam, ab11973, 1:200 dilution), PARP1 (Santacruz, sc-8007, 1:100 dilution), NUCKS1 (Abcam, ab84710, 1:100 dilution), SMARCA5 (Atlas Antibodies, HPA008751, 1:200 dilution), NF1B (Abcam, ab186738, 1:150 dilution), PTBP1 (Abcam, ab5642, 1:100 dilution), YBX1 (Abcam, ab12148, 1:500 dilution), EGFR (Atlas antibodies, HPA018530, 1:100 dilution), MAPK1 (Abcam, ab32081, 1:100 dilution), CD44 (BioGenex, AM310-5M, Ready to use), SOX2 (Invitrogen, PA-1-16968, 1:150 dilution), TNC (Cloud-clone corp, MAB975Hu22, 1:100 dilution) and MMP13 (Abcam, ab39012, 1:100 dilution) followed by peroxidase-labeled polymer conjugate to anti-goat, anti-mouse or anti-rabbit immunoglobulins compatible with the primary antibody for 1 h. After washing, sections were incubated for 5–20 min. with the 3, 3 - diaminobenzidine chromogen (DAB) system to develop the stain. .. Sections were counter stained with Mayer’s hematoxylin for about 30–60 sec.

    Quantitative RT-PCR:

    Article Title: Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Peripherin Abcam ab4666; RRID: AB_449340 MAP2 Abcam ab5392; RRID: AB_2138153 NEFH Abcam ab72996; RRID: AB_2149618 TUJ1 (mouse) Abcam ab78078; RRID: AB_2256751 TUJ1 (chicken) Abcam ab107216; RRID: AB_10899689 NeuN Abcam ab104225; RRID: AB_10711153 BRN3A Millipore MAB1585; RRID: AB_94166 ISL1 DSHB 40.2D6; RRID: AB_528315 TRKA Abcam ab76291; RRID: AB_1524514 TRKB Alomone Labs ANT-019-AG; RRID: AB_10917156 TRKC OriGene TA336908 OCT4 Abcam ab19857; RRID: AB_445175 SSEA4 STEMCELL Technologies 60062 NANOG ReproCELL RCAB0004P-F; RRID: AB_567470 TRA-1-81 Millipore MAB4381; RRID: AB_177638 SOX2 Thermo Fisher PA1-16968; RRID: AB_2195781 TRA-1-60 Millipore MAB4360; RRID: AB_2119183 Chemicals, Peptides, and Recombinant Proteins Y-27632 Tocris 1254 Doxycycline Clontech 631311 BDNF R Systems 248BD025 GDNF R Systems 212GD010 b-NGF R Systems 256GF100 NT-3 R Systems 267N3025 bFGF R Systems 233FB025 EGF R Systems 236EG200 SB431542 Tocris 1614 Retinoic acid Sigma-Aldrich R2625 AITC Sigma-Aldrich 377430 Capsaicin Sigma-Aldrich M2028 Menthol Sigma-Aldrich M2772 a,b-meATP Sigma-Aldrich M6517 RQ-00203078 Sigma-Aldrich SML1602 Tetrodotoxin Tocris 1078 Critical Commercial Assays Taqman Gene Expression Assay Thermo Fisher 18080085 Taqman RT-qPCR probes (see Oligonucleotides below) Thermo Fisher N/A RNAscope Fluorescent Multiplex Detection Reagents ACD 320851 RNAscope Probe – Hs-TRPM8 ACD 543121 RNAscope Probe – Hs-TRPM8-C3 ACD 543121-C3 RNAscope Probe – Hs-PIEZO2 ACD 449951 RNAscope Probe – Hs-PIEZO2-C2 ACD 449951-C2 RNAscope Probe – Hs-NTRK1 ACD 402631 (Continued on next page) Cell Reports 30, 932–946.e1–e7, January 21, 2020 e1 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER RNAscope Probe – Hs-NTRK2-C2 ACD 402621-C2 RNAscope Probe – Hs-NTRK3-C3 ACD 406341-C3 RNAscope Probe – Hs-TUBB3-C2 ACD 318901-C2 Deposited Data Bulk RNA sequencing data https://www.ncbi.nlm. nih.gov/geo/ Accession GSE139273 Single-nuclei RNA sequencing data https://www.ncbi.nlm. nih.gov/geo/ Accession GSE139409 Experimental Models: Cell Lines WTC11 iPSC Miyaoka et al., 2014 Coriell GM25256 WTC11 NGN2 iPSC Wang et al., 2017 N/A WTC11 NGN2-BRN3A iPSC (C1) This study N/A WTC11 NGN2-BRN3A iPSC, PIEZO2 knockout (KO) This study N/A PIEZO2 patient 1 NGN2-BRN3A iPSC (P1) This study N/A PIEZO2 patient 1 healthy sibling NGN2-BRN3A iPSC (C2) This study N/A PIEZO2 patient 2 NGN2-BRN3A iPSC (P2) This study N/A PIEZO2 patient 2 NGN2-BRN3A iPSC, CRISPR-Cas9-corrected (P2C) This study N/A Oligonucleotides CLYBL wild type genotyping primer, forward: 50-TGACTAAACACTGT GCCCCA-30 This study N/A CLYBL wild type genotyping primer, reverse: 50-AGGCAGGATGAAT TGGTGGA-30 This study N/A CLYBL 50 insert genotyping primer, forward: 50-CAGACAAGTCAGT AGGGCCA-30 This study N/A CLYBL 50 insert genotyping primer, reverse: 50-AGAAGACTTCCTCT GCCCTC-30 This study N/A CLYBL 30 insert genotyping primer, forward: 50-GGTAGGAAGTGGT ACGGAAA-30 This study N/A CLYBL 30 insert genotyping primer, reverse: 50-GAACGATTTACTGG GCAGTC-30 This study N/A NGN2-BRN3A transgene RT-PCR primer, forward: 50-CAAGATC GAAACCCTGAGAT-30 This study N/A NGN2-BRN3A transgene RT-PCR primer, reverse: 50-GATGGGTAC TTATGCTCAGG-30 This study N/A PIEZO2 exon 32 sequencing primer, forward: 50-AGACCCAATAT GCCAACACC-30 Chesler et al., 2016 N/A PIEZO2 exon 32 sequencing primer, reverse: 50-GGGAGCAGGCA TCATTACAA-30 Chesler et al., 2016 N/A PIEZO2 exon 35 sequencing primer, forward: 50-GGTAAAACATC GCTGGGCTA-30 Chesler et al., 2016 N/A PIEZO2 exon 35 sequencing primer, reverse: 50-AAGGGGTTATG CCACAACTG-30 Chesler et al., 2016 N/A RT-qPCR primer - RPLP0 IDT Hs.PT.39a.22214824 RT-qPCR primer - ISL1 Thermo Fisher Hs00158126_m1 RT-qPCR primer - POU4F1 Thermo Fisher Hs00366711_m1 RT-qPCR primer - NTRK1 Thermo Fisher Hs01021011_m1 RT-qPCR primer - NTRK2 Thermo Fisher Hs00178811_m1 RT-qPCR primer - PIEZO1 Thermo Fisher Hs00207230_m1 RT-qPCR primer - PIEZO2 Thermo Fisher Hs00926218_m1 RT-qPCR primer - SCN9A Thermo Fisher Hs00161567_m1 RT-qPCR primer - SCN10A Thermo Fisher Hs01045137_m1 (Continued on next page) e2 Cell Reports 30, 932–946.e1–e7, January 21, 2020

    Polyacrylamide Gel Electrophoresis:

    Article Title: Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Peripherin Abcam ab4666; RRID: AB_449340 MAP2 Abcam ab5392; RRID: AB_2138153 NEFH Abcam ab72996; RRID: AB_2149618 TUJ1 (mouse) Abcam ab78078; RRID: AB_2256751 TUJ1 (chicken) Abcam ab107216; RRID: AB_10899689 NeuN Abcam ab104225; RRID: AB_10711153 BRN3A Millipore MAB1585; RRID: AB_94166 ISL1 DSHB 40.2D6; RRID: AB_528315 TRKA Abcam ab76291; RRID: AB_1524514 TRKB Alomone Labs ANT-019-AG; RRID: AB_10917156 TRKC OriGene TA336908 OCT4 Abcam ab19857; RRID: AB_445175 SSEA4 STEMCELL Technologies 60062 NANOG ReproCELL RCAB0004P-F; RRID: AB_567470 TRA-1-81 Millipore MAB4381; RRID: AB_177638 SOX2 Thermo Fisher PA1-16968; RRID: AB_2195781 TRA-1-60 Millipore MAB4360; RRID: AB_2119183 Chemicals, Peptides, and Recombinant Proteins Y-27632 Tocris 1254 Doxycycline Clontech 631311 BDNF R Systems 248BD025 GDNF R Systems 212GD010 b-NGF R Systems 256GF100 NT-3 R Systems 267N3025 bFGF R Systems 233FB025 EGF R Systems 236EG200 SB431542 Tocris 1614 Retinoic acid Sigma-Aldrich R2625 AITC Sigma-Aldrich 377430 Capsaicin Sigma-Aldrich M2028 Menthol Sigma-Aldrich M2772 a,b-meATP Sigma-Aldrich M6517 RQ-00203078 Sigma-Aldrich SML1602 Tetrodotoxin Tocris 1078 Critical Commercial Assays Taqman Gene Expression Assay Thermo Fisher 18080085 Taqman RT-qPCR probes (see Oligonucleotides below) Thermo Fisher N/A RNAscope Fluorescent Multiplex Detection Reagents ACD 320851 RNAscope Probe – Hs-TRPM8 ACD 543121 RNAscope Probe – Hs-TRPM8-C3 ACD 543121-C3 RNAscope Probe – Hs-PIEZO2 ACD 449951 RNAscope Probe – Hs-PIEZO2-C2 ACD 449951-C2 RNAscope Probe – Hs-NTRK1 ACD 402631 (Continued on next page) Cell Reports 30, 932–946.e1–e7, January 21, 2020 e1 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER RNAscope Probe – Hs-NTRK2-C2 ACD 402621-C2 RNAscope Probe – Hs-NTRK3-C3 ACD 406341-C3 RNAscope Probe – Hs-TUBB3-C2 ACD 318901-C2 Deposited Data Bulk RNA sequencing data https://www.ncbi.nlm. nih.gov/geo/ Accession GSE139273 Single-nuclei RNA sequencing data https://www.ncbi.nlm. nih.gov/geo/ Accession GSE139409 Experimental Models: Cell Lines WTC11 iPSC Miyaoka et al., 2014 Coriell GM25256 WTC11 NGN2 iPSC Wang et al., 2017 N/A WTC11 NGN2-BRN3A iPSC (C1) This study N/A WTC11 NGN2-BRN3A iPSC, PIEZO2 knockout (KO) This study N/A PIEZO2 patient 1 NGN2-BRN3A iPSC (P1) This study N/A PIEZO2 patient 1 healthy sibling NGN2-BRN3A iPSC (C2) This study N/A PIEZO2 patient 2 NGN2-BRN3A iPSC (P2) This study N/A PIEZO2 patient 2 NGN2-BRN3A iPSC, CRISPR-Cas9-corrected (P2C) This study N/A Oligonucleotides CLYBL wild type genotyping primer, forward: 50-TGACTAAACACTGT GCCCCA-30 This study N/A CLYBL wild type genotyping primer, reverse: 50-AGGCAGGATGAAT TGGTGGA-30 This study N/A CLYBL 50 insert genotyping primer, forward: 50-CAGACAAGTCAGT AGGGCCA-30 This study N/A CLYBL 50 insert genotyping primer, reverse: 50-AGAAGACTTCCTCT GCCCTC-30 This study N/A CLYBL 30 insert genotyping primer, forward: 50-GGTAGGAAGTGGT ACGGAAA-30 This study N/A CLYBL 30 insert genotyping primer, reverse: 50-GAACGATTTACTGG GCAGTC-30 This study N/A NGN2-BRN3A transgene RT-PCR primer, forward: 50-CAAGATC GAAACCCTGAGAT-30 This study N/A NGN2-BRN3A transgene RT-PCR primer, reverse: 50-GATGGGTAC TTATGCTCAGG-30 This study N/A PIEZO2 exon 32 sequencing primer, forward: 50-AGACCCAATAT GCCAACACC-30 Chesler et al., 2016 N/A PIEZO2 exon 32 sequencing primer, reverse: 50-GGGAGCAGGCA TCATTACAA-30 Chesler et al., 2016 N/A PIEZO2 exon 35 sequencing primer, forward: 50-GGTAAAACATC GCTGGGCTA-30 Chesler et al., 2016 N/A PIEZO2 exon 35 sequencing primer, reverse: 50-AAGGGGTTATG CCACAACTG-30 Chesler et al., 2016 N/A RT-qPCR primer - RPLP0 IDT Hs.PT.39a.22214824 RT-qPCR primer - ISL1 Thermo Fisher Hs00158126_m1 RT-qPCR primer - POU4F1 Thermo Fisher Hs00366711_m1 RT-qPCR primer - NTRK1 Thermo Fisher Hs01021011_m1 RT-qPCR primer - NTRK2 Thermo Fisher Hs00178811_m1 RT-qPCR primer - PIEZO1 Thermo Fisher Hs00207230_m1 RT-qPCR primer - PIEZO2 Thermo Fisher Hs00926218_m1 RT-qPCR primer - SCN9A Thermo Fisher Hs00161567_m1 RT-qPCR primer - SCN10A Thermo Fisher Hs01045137_m1 (Continued on next page) e2 Cell Reports 30, 932–946.e1–e7, January 21, 2020

    Staining:

    Article Title: Human Stem/Progenitor Cells from Bone Marrow Enhance Glial Differentiation of Rat Neural Stem Cells: A Role for Transforming Growth Factor ? and Notch Signaling
    Article Snippet: .. Slides were subsequently incubated over night at 4°C with the following primary antibodies diluted in blocking buffer: Mouse antinestin (clone 401; BD Bioscience, cat# 556309; 1:1,000) and rabbit anti-Sox2 (Thermo Scientific, cat# PA1-16968; 1:250) were used to stain neural stem/progenitors; rabbit anti-Ki67 (Novocastra, cat# NSL-Ki67p; 1:8,000) to stain proliferating cells; rabbit anti-glial fibrillary acidic protein (GFAP; Chemicon, cat# AB5804; 1:1,000 or Sigma-Aldrich, cat# G9269; 1:1,000) or mouse anti-GFAP (clone GA-5; Cell Signaling, cat# 3670; 1:500) to stain astrocytes; mouse anti-oligodendrocytes (RIP, clone NSC-1; Chemicon, cat# MAB1580; 1:20,000) and mouse anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (2′,3′-cyclic nucleotide 3′-phospho-diesterase [CNPase], clone 11-5B; Sigma-Aldrich, cat# C5922; 1:100) to stain oligodendrocytes; mouse anti-β-III tubulin (TUJ1; Covance, Cat# MMS-435P; 1:1,000) or rabbit anti-β-III tubulin (Abcam, cat# AB18207; 1:2,000) to stain neurons; rabbit anti-Jagged 1 (Abcam, cat#Ab7771; 1:200) and goat anti-Notch 1 (R & D Systems, cat# AF1057; 1:100) to stain Notch signaling proteins. .. After PBS washes, coverslips were incubated with flourescent dye-conjugated secondary antibodies for 1 h at room temperature.

    Article Title: Altered transcriptional regulatory proteins in glioblastoma and YBX1 as a potential regulator of tumor invasion
    Article Snippet: .. Sections were then incubated for 1.5 h at room temperature with primary antibodies - HMGB2 (Abcam, ab11973, 1:200 dilution), PARP1 (Santacruz, sc-8007, 1:100 dilution), NUCKS1 (Abcam, ab84710, 1:100 dilution), SMARCA5 (Atlas Antibodies, HPA008751, 1:200 dilution), NF1B (Abcam, ab186738, 1:150 dilution), PTBP1 (Abcam, ab5642, 1:100 dilution), YBX1 (Abcam, ab12148, 1:500 dilution), EGFR (Atlas antibodies, HPA018530, 1:100 dilution), MAPK1 (Abcam, ab32081, 1:100 dilution), CD44 (BioGenex, AM310-5M, Ready to use), SOX2 (Invitrogen, PA-1-16968, 1:150 dilution), TNC (Cloud-clone corp, MAB975Hu22, 1:100 dilution) and MMP13 (Abcam, ab39012, 1:100 dilution) followed by peroxidase-labeled polymer conjugate to anti-goat, anti-mouse or anti-rabbit immunoglobulins compatible with the primary antibody for 1 h. After washing, sections were incubated for 5–20 min. with the 3, 3 - diaminobenzidine chromogen (DAB) system to develop the stain. .. Sections were counter stained with Mayer’s hematoxylin for about 30–60 sec.

    Western Blot:

    Article Title: Akt Protein Kinase, miR-200/miR-182 Expression and Epithelial-Mesenchymal Transition Proteins in Hibernating Ground Squirrels
    Article Snippet: .. The following antibodies were used for the Western blot analyses: anti-E-CDH (1:500, Abcam), anti-ZO-1 (1:500, Cell Signaling), anti-Zeb1 (1:250, Santa Cruz), anti-P-CDH (1:500, BD Biosciences), anti-Vimentin (1:1000, Abcam), anti-Snai1 (1:500, Cell Signaling), anti-N-Cadherin (anti-N-CDH; 1:500, Santa Cruz), anti-Caveolin (1:1000, Cell Signaling), anti-Sox-2 (1:500, ThermoFisher), anti-β–actin (1:10,000, Sigma). .. The following antibodies from Cell Signaling Technology were used at 1:1000 dilutions: anti-Akt1, Akt2, p-Akt (S473), p-Akt2 (S-474), p-Akt (T308), GSK3β (total), p-GSK3β (S9), PRAS40 (total) and p-PRAS40 (T246).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Human Stem/Progenitor Cells from Bone Marrow Enhance Glial Differentiation of Rat Neural Stem Cells: A Role for Transforming Growth Factor ? and Notch Signaling
    Article Snippet: .. Slides were subsequently incubated over night at 4°C with the following primary antibodies diluted in blocking buffer: Mouse antinestin (clone 401; BD Bioscience, cat# 556309; 1:1,000) and rabbit anti-Sox2 (Thermo Scientific, cat# PA1-16968; 1:250) were used to stain neural stem/progenitors; rabbit anti-Ki67 (Novocastra, cat# NSL-Ki67p; 1:8,000) to stain proliferating cells; rabbit anti-glial fibrillary acidic protein (GFAP; Chemicon, cat# AB5804; 1:1,000 or Sigma-Aldrich, cat# G9269; 1:1,000) or mouse anti-GFAP (clone GA-5; Cell Signaling, cat# 3670; 1:500) to stain astrocytes; mouse anti-oligodendrocytes (RIP, clone NSC-1; Chemicon, cat# MAB1580; 1:20,000) and mouse anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (2′,3′-cyclic nucleotide 3′-phospho-diesterase [CNPase], clone 11-5B; Sigma-Aldrich, cat# C5922; 1:100) to stain oligodendrocytes; mouse anti-β-III tubulin (TUJ1; Covance, Cat# MMS-435P; 1:1,000) or rabbit anti-β-III tubulin (Abcam, cat# AB18207; 1:2,000) to stain neurons; rabbit anti-Jagged 1 (Abcam, catAb7771; 1:200) and goat anti-Notch 1 (R & D Systems, cat# AF1057; 1:100) to stain Notch signaling proteins. .. After PBS washes, coverslips were incubated with flourescent dye-conjugated secondary antibodies for 1 h at room temperature.

    Recombinant:

    Article Title: Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Peripherin Abcam ab4666; RRID: AB_449340 MAP2 Abcam ab5392; RRID: AB_2138153 NEFH Abcam ab72996; RRID: AB_2149618 TUJ1 (mouse) Abcam ab78078; RRID: AB_2256751 TUJ1 (chicken) Abcam ab107216; RRID: AB_10899689 NeuN Abcam ab104225; RRID: AB_10711153 BRN3A Millipore MAB1585; RRID: AB_94166 ISL1 DSHB 40.2D6; RRID: AB_528315 TRKA Abcam ab76291; RRID: AB_1524514 TRKB Alomone Labs ANT-019-AG; RRID: AB_10917156 TRKC OriGene TA336908 OCT4 Abcam ab19857; RRID: AB_445175 SSEA4 STEMCELL Technologies 60062 NANOG ReproCELL RCAB0004P-F; RRID: AB_567470 TRA-1-81 Millipore MAB4381; RRID: AB_177638 SOX2 Thermo Fisher PA1-16968; RRID: AB_2195781 TRA-1-60 Millipore MAB4360; RRID: AB_2119183 Chemicals, Peptides, and Recombinant Proteins Y-27632 Tocris 1254 Doxycycline Clontech 631311 BDNF R Systems 248BD025 GDNF R Systems 212GD010 b-NGF R Systems 256GF100 NT-3 R Systems 267N3025 bFGF R Systems 233FB025 EGF R Systems 236EG200 SB431542 Tocris 1614 Retinoic acid Sigma-Aldrich R2625 AITC Sigma-Aldrich 377430 Capsaicin Sigma-Aldrich M2028 Menthol Sigma-Aldrich M2772 a,b-meATP Sigma-Aldrich M6517 RQ-00203078 Sigma-Aldrich SML1602 Tetrodotoxin Tocris 1078 Critical Commercial Assays Taqman Gene Expression Assay Thermo Fisher 18080085 Taqman RT-qPCR probes (see Oligonucleotides below) Thermo Fisher N/A RNAscope Fluorescent Multiplex Detection Reagents ACD 320851 RNAscope Probe – Hs-TRPM8 ACD 543121 RNAscope Probe – Hs-TRPM8-C3 ACD 543121-C3 RNAscope Probe – Hs-PIEZO2 ACD 449951 RNAscope Probe – Hs-PIEZO2-C2 ACD 449951-C2 RNAscope Probe – Hs-NTRK1 ACD 402631 (Continued on next page) Cell Reports 30, 932–946.e1–e7, January 21, 2020 e1 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER RNAscope Probe – Hs-NTRK2-C2 ACD 402621-C2 RNAscope Probe – Hs-NTRK3-C3 ACD 406341-C3 RNAscope Probe – Hs-TUBB3-C2 ACD 318901-C2 Deposited Data Bulk RNA sequencing data https://www.ncbi.nlm. nih.gov/geo/ Accession GSE139273 Single-nuclei RNA sequencing data https://www.ncbi.nlm. nih.gov/geo/ Accession GSE139409 Experimental Models: Cell Lines WTC11 iPSC Miyaoka et al., 2014 Coriell GM25256 WTC11 NGN2 iPSC Wang et al., 2017 N/A WTC11 NGN2-BRN3A iPSC (C1) This study N/A WTC11 NGN2-BRN3A iPSC, PIEZO2 knockout (KO) This study N/A PIEZO2 patient 1 NGN2-BRN3A iPSC (P1) This study N/A PIEZO2 patient 1 healthy sibling NGN2-BRN3A iPSC (C2) This study N/A PIEZO2 patient 2 NGN2-BRN3A iPSC (P2) This study N/A PIEZO2 patient 2 NGN2-BRN3A iPSC, CRISPR-Cas9-corrected (P2C) This study N/A Oligonucleotides CLYBL wild type genotyping primer, forward: 50-TGACTAAACACTGT GCCCCA-30 This study N/A CLYBL wild type genotyping primer, reverse: 50-AGGCAGGATGAAT TGGTGGA-30 This study N/A CLYBL 50 insert genotyping primer, forward: 50-CAGACAAGTCAGT AGGGCCA-30 This study N/A CLYBL 50 insert genotyping primer, reverse: 50-AGAAGACTTCCTCT GCCCTC-30 This study N/A CLYBL 30 insert genotyping primer, forward: 50-GGTAGGAAGTGGT ACGGAAA-30 This study N/A CLYBL 30 insert genotyping primer, reverse: 50-GAACGATTTACTGG GCAGTC-30 This study N/A NGN2-BRN3A transgene RT-PCR primer, forward: 50-CAAGATC GAAACCCTGAGAT-30 This study N/A NGN2-BRN3A transgene RT-PCR primer, reverse: 50-GATGGGTAC TTATGCTCAGG-30 This study N/A PIEZO2 exon 32 sequencing primer, forward: 50-AGACCCAATAT GCCAACACC-30 Chesler et al., 2016 N/A PIEZO2 exon 32 sequencing primer, reverse: 50-GGGAGCAGGCA TCATTACAA-30 Chesler et al., 2016 N/A PIEZO2 exon 35 sequencing primer, forward: 50-GGTAAAACATC GCTGGGCTA-30 Chesler et al., 2016 N/A PIEZO2 exon 35 sequencing primer, reverse: 50-AAGGGGTTATG CCACAACTG-30 Chesler et al., 2016 N/A RT-qPCR primer - RPLP0 IDT Hs.PT.39a.22214824 RT-qPCR primer - ISL1 Thermo Fisher Hs00158126_m1 RT-qPCR primer - POU4F1 Thermo Fisher Hs00366711_m1 RT-qPCR primer - NTRK1 Thermo Fisher Hs01021011_m1 RT-qPCR primer - NTRK2 Thermo Fisher Hs00178811_m1 RT-qPCR primer - PIEZO1 Thermo Fisher Hs00207230_m1 RT-qPCR primer - PIEZO2 Thermo Fisher Hs00926218_m1 RT-qPCR primer - SCN9A Thermo Fisher Hs00161567_m1 RT-qPCR primer - SCN10A Thermo Fisher Hs01045137_m1 (Continued on next page) e2 Cell Reports 30, 932–946.e1–e7, January 21, 2020

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    Thermo Fisher rabbit anti sox2
    Genome Stability and Pluripotency of Corrected iPSC Lines (A and I) Phase-contrast images of USH2A -USH-iPSC clone B3B1 (A) and USH2A -RP-iPSC clone MS3F7 (I). (B and J) Genomic stability of USH2A -USH-iPSC B3B1 (B) and USH2A -RP-iPSC MS3F7 (J) as determined by a digital qPCR analysis of the most commonly rearranged regions reported in iPSCs. The copy number for each chromosomal position is shown with colored dots. (C–E and K–M) Pluripotency of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the markers OCT3/4 (C and K), <t>SOX2</t> (D and L), and NANOG (E and M), respectively. Scale bars, 50 μM. (F-H and N-P) Differentiation capacity of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the germ layer markers GFAP (ectoderm; F and N), SMA (mesoderm; G and O), and AFP (endoderm; H and P), respectively. Scale bars, 20 μM.
    Rabbit Anti Sox2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genome Stability and Pluripotency of Corrected iPSC Lines (A and I) Phase-contrast images of USH2A -USH-iPSC clone B3B1 (A) and USH2A -RP-iPSC clone MS3F7 (I). (B and J) Genomic stability of USH2A -USH-iPSC B3B1 (B) and USH2A -RP-iPSC MS3F7 (J) as determined by a digital qPCR analysis of the most commonly rearranged regions reported in iPSCs. The copy number for each chromosomal position is shown with colored dots. (C–E and K–M) Pluripotency of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the markers OCT3/4 (C and K), SOX2 (D and L), and NANOG (E and M), respectively. Scale bars, 50 μM. (F-H and N-P) Differentiation capacity of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the germ layer markers GFAP (ectoderm; F and N), SMA (mesoderm; G and O), and AFP (endoderm; H and P), respectively. Scale bars, 20 μM.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Genome Editing in Patient iPSCs Corrects the Most Prevalent USH2A Mutations and Reveals Intriguing Mutant mRNA Expression Profiles

    doi: 10.1016/j.omtm.2019.11.016

    Figure Lengend Snippet: Genome Stability and Pluripotency of Corrected iPSC Lines (A and I) Phase-contrast images of USH2A -USH-iPSC clone B3B1 (A) and USH2A -RP-iPSC clone MS3F7 (I). (B and J) Genomic stability of USH2A -USH-iPSC B3B1 (B) and USH2A -RP-iPSC MS3F7 (J) as determined by a digital qPCR analysis of the most commonly rearranged regions reported in iPSCs. The copy number for each chromosomal position is shown with colored dots. (C–E and K–M) Pluripotency of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the markers OCT3/4 (C and K), SOX2 (D and L), and NANOG (E and M), respectively. Scale bars, 50 μM. (F-H and N-P) Differentiation capacity of USH2A -USH-iPSC clone B3B1 and USH2A -RP-iPSC clone MS3F7 as determined by immunostaining of the germ layer markers GFAP (ectoderm; F and N), SMA (mesoderm; G and O), and AFP (endoderm; H and P), respectively. Scale bars, 20 μM.

    Article Snippet: Primary antibodies were used at a 1:200 dilution in blocking solution and incubated overnight at 4°C: rabbit anti-NANOG (Abcam, Paris, France), mouse anti-OCT3/4 (Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit anti-SOX2 (Thermo Fisher Scientific) for the iPSCs, and rabbit anti-GFAP (Dako, Les Ulis, France), mouse anti-SMA (Dako), and mouse anti-AFP (Sigma Aldrich) for the embryoid bodies.

    Techniques: Real-time Polymerase Chain Reaction, Immunostaining

    Expression of Schwann cell markers in peripheral nerves of Sox10 ΔeSC embryos. (A–L) Immunohistochemistry (A–J) and in situ hybridizations (K and L) were performed on transverse sections of wild-type (wt; A, C, E, G, I, and K) and Sox10 ΔeSC (B, D, F, H, J, and L) sciatic nerves at 16.5 (A, B, G, and H) and 18.5 dpc (C–F and I–L) using antibodies directed against Sox2 (A–D), Krox20 (E and F), Oct6 (G–J), and an antisense riboprobe for Mbp (K and L). (C, F, H, and J) Dotted lines indicate the circumference of the sciatic nerve. Bars, 50 µm.

    Journal: The Journal of Cell Biology

    Article Title: Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage

    doi: 10.1083/jcb.200912142

    Figure Lengend Snippet: Expression of Schwann cell markers in peripheral nerves of Sox10 ΔeSC embryos. (A–L) Immunohistochemistry (A–J) and in situ hybridizations (K and L) were performed on transverse sections of wild-type (wt; A, C, E, G, I, and K) and Sox10 ΔeSC (B, D, F, H, J, and L) sciatic nerves at 16.5 (A, B, G, and H) and 18.5 dpc (C–F and I–L) using antibodies directed against Sox2 (A–D), Krox20 (E and F), Oct6 (G–J), and an antisense riboprobe for Mbp (K and L). (C, F, H, and J) Dotted lines indicate the circumference of the sciatic nerve. Bars, 50 µm.

    Article Snippet: For immunohistochemistry, the following primary antibodies were used in various combinations: anti-Sox10 guinea pig antiserum (1:1,000; ), anti-Oct6 rabbit antiserum (1:2,000; ), anti-Krox20 rabbit antiserum (1:200; Covance), anti-Sox2 rabbit antiserum (1:500), anti-Ki67 rabbit antiserum (1:500; Thermo Fisher Scientific), anti-CD3 rabbit antiserum (1:500; Abcam), anti-GFP rabbit antiserum (1:500; Invitrogen), anti-Iba1 rabbit antiserum (1:250; Wako Chemicals USA, Inc.) antidesmin rabbit antiserum (1:1,000; Abcam), anti–von Willebrand factor rabbit antiserum (1:800; Abcam), and anti-PECAM rat antiserum (1:200; BD).

    Techniques: Expressing, Immunohistochemistry, In Situ

    Postnatal development of Sox10 ΔeSC mice and their sciatic nerves. (A) Weight of wild-type (open bars) and age-matched Sox10 ΔeSC (closed bars) mice was monitored at P8, P16, P24, and P32. Data are shown as means ± SEM ( n ≥ 6 for each genotype). Statistically significant differences from wild-type controls were observed from P16 onwards (**, P ≤ 0.01; and ***, P ≤ 0.001 by Student’s t test). (B) Macroscopic appearance of P32 sciatic nerves from wild-type (wt) and Sox10 ΔeSC mice. (C) Sciatic nerve thickness was quantified for wild-type and Sox10 ΔeSC mice at P8, P16, P24, and P32 by determining the area on proximal nerve sections. At least 20 sections from three mice were used per genotype for quantification. Data are presented as mean ± SEM. Differences were statistically significant between wild type and Sox10 ΔeSC mutant from P8 onwards as determined by Student’s t test (**, P ≤ 0.01; ***, P ≤ 0.001). (D–K) Immunohistochemistry was performed on sciatic nerve sections from wild-type (D, F, H, and J) and Sox10 ΔeSC (E, G, I, and K) mice at P16 (D, E, H, and I) and P32 (F, G, J, and K) using antibodies directed against Sox2 (D–G) and Krox20 (H–K). (D, F, I, and K) Dotted lines indicate the circumference of the sciatic nerve. (L–S) In situ hybridization was performed on sciatic nerve sections from wild-type (L, N, P, and R) and Sox10 ΔeSC (M, O, Q, and S) mice at P16 (L, M, P, and Q) and P32 (N, O, R, and S) using antisense riboprobes for Mpz (L–O) and Mbp (P–S). (T–W) Myelin sheaths were visualized by PPD staining of sciatic nerve sections from wild-type (T and V) and Sox10 ΔeSC (U and W) mice at P16 (T and U) and P32 (V and W). (X and Y) Electrophysiology on sciatic nerves of Sox10 ΔeSC mice. Compound action potentials were monopolarly recorded from isolated sciatic nerves of wild-type and Sox10 ΔeSC mice ( n = 2 each). Experiments were performed on both nerves of each animal with identical results within each genotype. Representative superimposed traces are presented for both genotypes showing fast nerve conduction along myelinated fibers (X) and slow conduction along nonmyelinated fibers (Y). The arrows point to components of different conduction velocities (meters/second). Bars: (B) 1 mm; (D–S) 50 µm; (T–W) 3 µm.

    Journal: The Journal of Cell Biology

    Article Title: Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage

    doi: 10.1083/jcb.200912142

    Figure Lengend Snippet: Postnatal development of Sox10 ΔeSC mice and their sciatic nerves. (A) Weight of wild-type (open bars) and age-matched Sox10 ΔeSC (closed bars) mice was monitored at P8, P16, P24, and P32. Data are shown as means ± SEM ( n ≥ 6 for each genotype). Statistically significant differences from wild-type controls were observed from P16 onwards (**, P ≤ 0.01; and ***, P ≤ 0.001 by Student’s t test). (B) Macroscopic appearance of P32 sciatic nerves from wild-type (wt) and Sox10 ΔeSC mice. (C) Sciatic nerve thickness was quantified for wild-type and Sox10 ΔeSC mice at P8, P16, P24, and P32 by determining the area on proximal nerve sections. At least 20 sections from three mice were used per genotype for quantification. Data are presented as mean ± SEM. Differences were statistically significant between wild type and Sox10 ΔeSC mutant from P8 onwards as determined by Student’s t test (**, P ≤ 0.01; ***, P ≤ 0.001). (D–K) Immunohistochemistry was performed on sciatic nerve sections from wild-type (D, F, H, and J) and Sox10 ΔeSC (E, G, I, and K) mice at P16 (D, E, H, and I) and P32 (F, G, J, and K) using antibodies directed against Sox2 (D–G) and Krox20 (H–K). (D, F, I, and K) Dotted lines indicate the circumference of the sciatic nerve. (L–S) In situ hybridization was performed on sciatic nerve sections from wild-type (L, N, P, and R) and Sox10 ΔeSC (M, O, Q, and S) mice at P16 (L, M, P, and Q) and P32 (N, O, R, and S) using antisense riboprobes for Mpz (L–O) and Mbp (P–S). (T–W) Myelin sheaths were visualized by PPD staining of sciatic nerve sections from wild-type (T and V) and Sox10 ΔeSC (U and W) mice at P16 (T and U) and P32 (V and W). (X and Y) Electrophysiology on sciatic nerves of Sox10 ΔeSC mice. Compound action potentials were monopolarly recorded from isolated sciatic nerves of wild-type and Sox10 ΔeSC mice ( n = 2 each). Experiments were performed on both nerves of each animal with identical results within each genotype. Representative superimposed traces are presented for both genotypes showing fast nerve conduction along myelinated fibers (X) and slow conduction along nonmyelinated fibers (Y). The arrows point to components of different conduction velocities (meters/second). Bars: (B) 1 mm; (D–S) 50 µm; (T–W) 3 µm.

    Article Snippet: For immunohistochemistry, the following primary antibodies were used in various combinations: anti-Sox10 guinea pig antiserum (1:1,000; ), anti-Oct6 rabbit antiserum (1:2,000; ), anti-Krox20 rabbit antiserum (1:200; Covance), anti-Sox2 rabbit antiserum (1:500), anti-Ki67 rabbit antiserum (1:500; Thermo Fisher Scientific), anti-CD3 rabbit antiserum (1:500; Abcam), anti-GFP rabbit antiserum (1:500; Invitrogen), anti-Iba1 rabbit antiserum (1:250; Wako Chemicals USA, Inc.) antidesmin rabbit antiserum (1:1,000; Abcam), anti–von Willebrand factor rabbit antiserum (1:800; Abcam), and anti-PECAM rat antiserum (1:200; BD).

    Techniques: Mouse Assay, Mutagenesis, Immunohistochemistry, In Situ Hybridization, Staining, Isolation

    Schwann cell proliferation in sciatic nerves of Sox10 ΔeSC mice. (A and B) The absolute number of Sox2-positive cells was determined in sciatic nerves of Sox10 ΔeSC mice at P8, P16, P24, and P32 (A) and set in relation to the total number of cells (B). (C) Cell numbers were also determined for YFP-expressing cells in sciatic nerves of Dhh::Cre, Rosa26 stopfloxYFP ( Rosa26 DhhYFP ; open bars) and Dhh::Cre, Sox10 fl/fl , Rosa26 stopfloxYFP mice ( Sox10 ΔeSC , Rosa26 DhhYFP ; closed bars) at P8, P16, P24, and P32. (D–G) Coimmunohistochemistry was performed on sciatic nerve sections of Sox10 ΔeSC , Rosa26 DhhYFP mice at P8 (D), P16 (E), P24 (F), and P32 (G) using antibodies against Sox2 (red) and YFP (green). (H) Proliferation rates of wild-type (wt) and Sox10 ΔeSC Schwann cells were determined at P8, P16, P24, and P32 by determining the fraction of Ki67-positive cells among the YFP-labeled cells in Rosa26 DhhYFP (open bars) and Sox10 ΔeSC , Rosa26 DhhYFP mice (closed bars). (I) The proliferation rates of Sox10 ΔeSC Schwann cells (see H) were also used to determine the relative contribution of Schwann cells to the overall proliferation in sciatic nerves of Sox10 ΔeSC mice. For all quantifications, at least 20 sections from three mice were used per genotype. Data are presented as mean ± SEM. According to the Student’s t test, differences were statistically significant between wild type and Sox10 ΔeSC mutant as indicated (**, P ≤ 0.01; ***, P ≤ 0.001). Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage

    doi: 10.1083/jcb.200912142

    Figure Lengend Snippet: Schwann cell proliferation in sciatic nerves of Sox10 ΔeSC mice. (A and B) The absolute number of Sox2-positive cells was determined in sciatic nerves of Sox10 ΔeSC mice at P8, P16, P24, and P32 (A) and set in relation to the total number of cells (B). (C) Cell numbers were also determined for YFP-expressing cells in sciatic nerves of Dhh::Cre, Rosa26 stopfloxYFP ( Rosa26 DhhYFP ; open bars) and Dhh::Cre, Sox10 fl/fl , Rosa26 stopfloxYFP mice ( Sox10 ΔeSC , Rosa26 DhhYFP ; closed bars) at P8, P16, P24, and P32. (D–G) Coimmunohistochemistry was performed on sciatic nerve sections of Sox10 ΔeSC , Rosa26 DhhYFP mice at P8 (D), P16 (E), P24 (F), and P32 (G) using antibodies against Sox2 (red) and YFP (green). (H) Proliferation rates of wild-type (wt) and Sox10 ΔeSC Schwann cells were determined at P8, P16, P24, and P32 by determining the fraction of Ki67-positive cells among the YFP-labeled cells in Rosa26 DhhYFP (open bars) and Sox10 ΔeSC , Rosa26 DhhYFP mice (closed bars). (I) The proliferation rates of Sox10 ΔeSC Schwann cells (see H) were also used to determine the relative contribution of Schwann cells to the overall proliferation in sciatic nerves of Sox10 ΔeSC mice. For all quantifications, at least 20 sections from three mice were used per genotype. Data are presented as mean ± SEM. According to the Student’s t test, differences were statistically significant between wild type and Sox10 ΔeSC mutant as indicated (**, P ≤ 0.01; ***, P ≤ 0.001). Bars, 10 µm.

    Article Snippet: For immunohistochemistry, the following primary antibodies were used in various combinations: anti-Sox10 guinea pig antiserum (1:1,000; ), anti-Oct6 rabbit antiserum (1:2,000; ), anti-Krox20 rabbit antiserum (1:200; Covance), anti-Sox2 rabbit antiserum (1:500), anti-Ki67 rabbit antiserum (1:500; Thermo Fisher Scientific), anti-CD3 rabbit antiserum (1:500; Abcam), anti-GFP rabbit antiserum (1:500; Invitrogen), anti-Iba1 rabbit antiserum (1:250; Wako Chemicals USA, Inc.) antidesmin rabbit antiserum (1:1,000; Abcam), anti–von Willebrand factor rabbit antiserum (1:800; Abcam), and anti-PECAM rat antiserum (1:200; BD).

    Techniques: Mouse Assay, Expressing, Labeling, Mutagenesis

    Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p

    Journal: Nature Communications

    Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

    doi: 10.1038/s41467-018-08140-7

    Figure Lengend Snippet: Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p

    Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

    Techniques: Cell Culture, Staining

    Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p

    Journal: Nature Communications

    Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

    doi: 10.1038/s41467-018-08140-7

    Figure Lengend Snippet: Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p

    Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

    Techniques: In Vitro, Cell Culture, Staining, Marker, Expressing, Two Tailed Test

    Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p

    Journal: Nature Communications

    Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

    doi: 10.1038/s41467-018-08140-7

    Figure Lengend Snippet: Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p

    Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

    Techniques: Cell Culture, Staining

    Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p

    Journal: Nature Communications

    Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

    doi: 10.1038/s41467-018-08140-7

    Figure Lengend Snippet: Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p

    Article Snippet: For analysis of murine tissue and cells, primary antibodies at working concentrations were: rat anti-BrdU (1:100; Accurate Chemical, cat# YSRTMCA2060GA), rat anti-CD11b (1:50; BD Biosciences, cat# 550282), mouse anti-CD45 (1:100, BioLegend, cat# 103101), rat anti-CD4 (1:200, BD Bioscience, cat# 550278) rabbit anti Chi3l3 (1:50; Stemcell Technology, cat# 60130), rabbit anti-Doublecortin (Dcx, 1:100; Abcam, cat# ab18723), mouse anti-GFAP (1:500; BD Bioscience, cat# 610566), mouse anti-Ki76-FITC (1:100; BD Bioscience, cat# 617472), mouse anti-Map2 (1:250; Sigma-Aldrich, cat# M9942), rabbit anti-NG2 (1:100; Millipore, cat# ab5320), mouse anti-O4 (1:100; Millipore, cat# MAB345), rabbit anti-p-Pyk2 (Tyr402; 1:500, CST, cat# 3291 S), rabbit anti-p-cRaf (Ser259; 1:500, CST, cat# 9421 P), rabbit anti-Sox2 (1:500, Thermo Fisher Scientific, cat# A24339), rabbit anti-p-p38MAPK (THr180/Tyr182; 1:500, CST, cat#4511 P), rabbit anti-p-PLCγ2 (Tyr1217; 1:500, CST, cat# 3871 P), rabbit anti-p-PI(3)K (Tyr458; 1:500, CST cat# 4228 P), rabbit anti-p-Erk1/2 (Thr202/Tyr204; 1:500, CST, cat# 9101), rabbit anti-p-EGFR (Tyr1068; 1:100, CST, cat# 3777).

    Techniques: In Vitro, Cell Culture, Staining, Marker, Expressing, Two Tailed Test