chicken anti-gfp Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher chicken anti gfp
    Effects of expression of full-length Cad7 and full-length Cad6b constructs in the chick hindbrain. ( A-L ) E4 hindbrains immunostained with <t>anti-Islet1/2</t> (red) and <t>anti-GFP</t> (green) antibodies to detect electroporated neurons. Hindbrains were electroporated
    Chicken Anti Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1495 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chicken anti gfp/product/Thermo Fisher
    Average 99 stars, based on 1495 article reviews
    Price from $9.99 to $1999.99
    chicken anti gfp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore chicken anti gfp
    Elevated SGG activity in s-LN v in rsk - mutants. (A) Analysis of the l-LN v and s-LN v arborization patterns in rsk - by staining for neuropeptide PDF expressed in s-LN v (excluding the 5 th LN v ) and l-LN v ) and ITP, which is restricted to the 5 th LN v and a single LN d ). Asterisks: ITP positive non-clock neurons. Scale bar 20 μm. (B) s-LN v in wild-type or rsk - mutants were identified by position and expression of a <t>GFP-reporter</t> under the control of clk 856 -Gal4 driver line. Brains were stained at ZT20 for GFP, <t>PER</t> and activated SGG (pY214-SGG). Pictures are representative examples from at least eight brains analyzed per genotype, recorded and processed in an identical manner. Scale bar: 5 μm. (C) Quantification of pY214-SGG signal intensities in s-LN v of rsk - ( n = 33 cells in eight brains) compared to wild-type ( n = 31 cells in eight brains). The pY214-SGG signal in each cell was first normalized to the corresponding GFP-signal and the combined values for wild-type were set to the arbitrary unit 1.
    Chicken Anti Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 854 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chicken anti gfp/product/Millipore
    Average 99 stars, based on 854 article reviews
    Price from $9.99 to $1999.99
    chicken anti gfp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Abcam chicken anti gfp
    Wnt signaling acts in the ECs to regulate <t>Tkv</t> expression. (A–F) Germaria with compromised Wnt signaling components (Arm [A and B, BL-35004] and Lgs [C, BL-37476]) or with overexpressed TCF DN (D and E) in ECs exhibit more spectrosome-containing cells and reduced tkv k16713 reporter expression. (F) Statistical data for spectrosome-containing cells in arm i or TCF DN expressing germarium. (G) A germarium containing ECs mutant for the pygo s123 allele (generated using the MARCM system and marked with an arrowhead) exhibits ectopic spectrosome-containing cells. Genotype: <t>hs-flp.UAS-CD8.GFP/+;tub-gal4/+;FRT82B.pygo</t> s123 /FRT82B.tub-gal80. (H) A pygo i (NIG-11518R) germarium harbors ectopic Dad-lacZ–positive spectrosome-containing cells. (I) A pygo i (NIG-11518R-1) germarium contains more pMad-positive spectrosome-containing cells. (J) A arm i (BL-35004) germarium expresses low levels of tkv transcripts (compared with Fig. 1 H ). (K) A lgs i (BL-37476) germarium expresses low levels of Tkv in the ECs (compared with Figure 1 I ). (L) One EC mutant for pygo s123 (arrowhead) exhibits reduced Tkv expression, compared with control EC (arrow) of the same germarium. Genotype: hs-flp;FRT82B.pygo s123 /FRT82B.ubi-GFP . (M–O) Forced expression of Tkv in lgs i (BL-37476) germaria strongly suppresses the formation of ectopic spectrosome-containing cells. ***, P
    Chicken Anti Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 6406 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chicken anti gfp/product/Abcam
    Average 94 stars, based on 6406 article reviews
    Price from $9.99 to $1999.99
    chicken anti gfp - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    93
    AvesLabs chicken anti gfp
    Expression of the FRET glucose sensor in neocortical slices. Representative single plane confocal image of a slice expressing the FLII12Pglu-700 μδ6 biosensor following sindbis viral transduction. Cells expressing the FRET glucose sensor are identified by their <t>GFP</t> fluorescence ( green ). Pyramidal cells are immunolabeled for the <t>Satb2</t> transcription factor ( red ). Dashed line represents layer I and II border. Note that most layer II and III transduced cells are stab2 positive.
    Chicken Anti Gfp, supplied by AvesLabs, used in various techniques. Bioz Stars score: 93/100, based on 2807 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chicken anti gfp/product/AvesLabs
    Average 93 stars, based on 2807 article reviews
    Price from $9.99 to $1999.99
    chicken anti gfp - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Abcam anti gfp antibody
    Characterization of In Vivo- Reprogrammed Cell Clusters in the GAs of <t>Nanog-GFP</t> and Pax3-GFP Mice (A) Nanog-GFP and Pax3-GFP transgenics were administered, 50 μg pOKS and 50 μg pM in 50 μL 0.9% saline, into the GA and dissected 2 days p.i. for histological analysis. (B) Clusters of reprogrammed cells were identified by H E and the green fluorescence resulting from either Nanog or Pax3 upregulation (100×; scale bars represent 50 μm). Bright-field and fluorescence images show the same region within the tissue. *p
    Anti Gfp Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 5138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gfp antibody/product/Abcam
    Average 99 stars, based on 5138 article reviews
    Price from $9.99 to $1999.99
    anti gfp antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher gfp tag polyclonal antibody
    Characterization of In Vivo- Reprogrammed Cell Clusters in the GAs of <t>Nanog-GFP</t> and Pax3-GFP Mice (A) Nanog-GFP and Pax3-GFP transgenics were administered, 50 μg pOKS and 50 μg pM in 50 μL 0.9% saline, into the GA and dissected 2 days p.i. for histological analysis. (B) Clusters of reprogrammed cells were identified by H E and the green fluorescence resulting from either Nanog or Pax3 upregulation (100×; scale bars represent 50 μm). Bright-field and fluorescence images show the same region within the tissue. *p
    Gfp Tag Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp tag polyclonal antibody/product/Thermo Fisher
    Average 99 stars, based on 8757 article reviews
    Price from $9.99 to $1999.99
    gfp tag polyclonal antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    90
    AvesLabs chicken polyclonal anti gfp
    Characterization of In Vivo- Reprogrammed Cell Clusters in the GAs of <t>Nanog-GFP</t> and Pax3-GFP Mice (A) Nanog-GFP and Pax3-GFP transgenics were administered, 50 μg pOKS and 50 μg pM in 50 μL 0.9% saline, into the GA and dissected 2 days p.i. for histological analysis. (B) Clusters of reprogrammed cells were identified by H E and the green fluorescence resulting from either Nanog or Pax3 upregulation (100×; scale bars represent 50 μm). Bright-field and fluorescence images show the same region within the tissue. *p
    Chicken Polyclonal Anti Gfp, supplied by AvesLabs, used in various techniques. Bioz Stars score: 90/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chicken polyclonal anti gfp/product/AvesLabs
    Average 90 stars, based on 125 article reviews
    Price from $9.99 to $1999.99
    chicken polyclonal anti gfp - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    88
    AvesLabs chicken anti green fluorescent protein gfp
    The embryonic POA gives rise to cells that migrate to the cortex. A , Schema of the experimental design followed in B–E′ . B , B′ , A representative case of the distribution of <t>GFP</t> ( B )- and Nkx2-1 ( B′ )-expressing cells in a coronal section through the telencephalon of an E15.5 embryo in which the POA was electroporated at E12.5. The white arrowhead indicates the location of basal forebrain cells derived from the POA. C , C′ , High-magnification images of the boxed areas shown in B and B′ , respectively. The open arrowheads in B and C mark labeled cells with migratory morphology in the subpallium and in the cortex. D , D′ , E , E′ , Images of representative cells found in the cortex. These cells typically stain for Calbindin ( D , D′ ) and do not express <t>Lhx6</t> ( E , E′ ). Scale bars: (in B ) B , B′ , 250 μm; (in C ) C , C′ , 100 μm; (in D ) D , D′ , E , E′ , 25 μm.
    Chicken Anti Green Fluorescent Protein Gfp, supplied by AvesLabs, used in various techniques. Bioz Stars score: 88/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chicken anti green fluorescent protein gfp/product/AvesLabs
    Average 88 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    chicken anti green fluorescent protein gfp - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    Image Search Results


    Effects of expression of full-length Cad7 and full-length Cad6b constructs in the chick hindbrain. ( A-L ) E4 hindbrains immunostained with anti-Islet1/2 (red) and anti-GFP (green) antibodies to detect electroporated neurons. Hindbrains were electroporated

    Journal: Development (Cambridge, England)

    Article Title: Cadherin-7 and cadherin-6B differentially regulate the growth, branching and guidance of cranial motor axons

    doi: 10.1242/dev.042457

    Figure Lengend Snippet: Effects of expression of full-length Cad7 and full-length Cad6b constructs in the chick hindbrain. ( A-L ) E4 hindbrains immunostained with anti-Islet1/2 (red) and anti-GFP (green) antibodies to detect electroporated neurons. Hindbrains were electroporated

    Article Snippet: Immunohistochemistry was performed on whole-mount or cryosections as described previously , using rabbit or chicken anti-GFP (1:1000; Invitrogen), mouse anti-Islet1/2 (4D5; 1:500), mouse anti-SC1 (1:100; DSHB), chicken anti-β-galactosidase, anti-sarcomeric myosin antibody MF20 (1:250; DSHB) and rabbit anti-phospho-Akt (1:500; Ab 9271, Cell Signaling Technologies).

    Techniques: Expressing, Construct

    egg is dispensable intrinsically for maintaining E-cadherin accumulation in the GSC-niche junction. (A) A GFP-negative marked egg mutant GSC (filled arrowhead) has similar E-cadherin accumulation in the GSC-niche junction to its neighboring GFP-positive unmarked control GSC (open arrowhead). The solid line highlights the boundary between the two GSCs. (B) GFP-negative marked egg follicle cells (filled arrowhead) have similar apical E-cadherin accumulation to their neighboring GFP-positive unmarked control follicle cells (open arrowhead). The solid line highlights the boundary between the mutant and control follicle cells. (C) nos-gal4 driven germ cell-specific expression of E-cadherin fails to rescue the stem cell loss phenotype of marked mutant egg GSCs. Scale bars: 10 µm.

    Journal: PLoS Genetics

    Article Title: Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

    doi: 10.1371/journal.pgen.1002426

    Figure Lengend Snippet: egg is dispensable intrinsically for maintaining E-cadherin accumulation in the GSC-niche junction. (A) A GFP-negative marked egg mutant GSC (filled arrowhead) has similar E-cadherin accumulation in the GSC-niche junction to its neighboring GFP-positive unmarked control GSC (open arrowhead). The solid line highlights the boundary between the two GSCs. (B) GFP-negative marked egg follicle cells (filled arrowhead) have similar apical E-cadherin accumulation to their neighboring GFP-positive unmarked control follicle cells (open arrowhead). The solid line highlights the boundary between the mutant and control follicle cells. (C) nos-gal4 driven germ cell-specific expression of E-cadherin fails to rescue the stem cell loss phenotype of marked mutant egg GSCs. Scale bars: 10 µm.

    Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Hts (1B1, 1∶4, DSHB), mouse anti-Lamin C (LC28.26, 1∶4, DSHB), mouse anti-Orb (4H8, 1∶4, DSHB), mouse anti-Fas3 (7G10, 1∶3, DSHB), rat anti-Vasa (1∶10, DSHB), rat anti-E-cadherin DCAD2 (1∶3, DSHB), polyclonal rabbit anti-GFP (1∶100, Molecular Probes), chicken anti-GFP antibody (1∶200, Invitrogen), rabbit anti-β-galactosidase (1∶100, Molecular Probes), rabbit anti-H3K9me3 (1∶200, Abcam ab8898) and rabbit anti-phosphorylated ERK1/2 (1∶200, a gift from Dr. Y. Cai).

    Techniques: Mutagenesis, Expressing

    Elevated SGG activity in s-LN v in rsk - mutants. (A) Analysis of the l-LN v and s-LN v arborization patterns in rsk - by staining for neuropeptide PDF expressed in s-LN v (excluding the 5 th LN v ) and l-LN v ) and ITP, which is restricted to the 5 th LN v and a single LN d ). Asterisks: ITP positive non-clock neurons. Scale bar 20 μm. (B) s-LN v in wild-type or rsk - mutants were identified by position and expression of a GFP-reporter under the control of clk 856 -Gal4 driver line. Brains were stained at ZT20 for GFP, PER and activated SGG (pY214-SGG). Pictures are representative examples from at least eight brains analyzed per genotype, recorded and processed in an identical manner. Scale bar: 5 μm. (C) Quantification of pY214-SGG signal intensities in s-LN v of rsk - ( n = 33 cells in eight brains) compared to wild-type ( n = 31 cells in eight brains). The pY214-SGG signal in each cell was first normalized to the corresponding GFP-signal and the combined values for wild-type were set to the arbitrary unit 1.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Drosophila RSK Influences the Pace of the Circadian Clock by Negative Regulation of Protein Kinase Shaggy Activity

    doi: 10.3389/fnmol.2018.00122

    Figure Lengend Snippet: Elevated SGG activity in s-LN v in rsk - mutants. (A) Analysis of the l-LN v and s-LN v arborization patterns in rsk - by staining for neuropeptide PDF expressed in s-LN v (excluding the 5 th LN v ) and l-LN v ) and ITP, which is restricted to the 5 th LN v and a single LN d ). Asterisks: ITP positive non-clock neurons. Scale bar 20 μm. (B) s-LN v in wild-type or rsk - mutants were identified by position and expression of a GFP-reporter under the control of clk 856 -Gal4 driver line. Brains were stained at ZT20 for GFP, PER and activated SGG (pY214-SGG). Pictures are representative examples from at least eight brains analyzed per genotype, recorded and processed in an identical manner. Scale bar: 5 μm. (C) Quantification of pY214-SGG signal intensities in s-LN v of rsk - ( n = 33 cells in eight brains) compared to wild-type ( n = 31 cells in eight brains). The pY214-SGG signal in each cell was first normalized to the corresponding GFP-signal and the combined values for wild-type were set to the arbitrary unit 1.

    Article Snippet: Dissected brains were fixed for 30 min at room temperature, then washed in PBT (PBS plus 0.3% Triton X-100, used for all washing steps) before blocking in PBT supplemented with 5% normal goat serum for 2 h. Incubation with the following primary antibodies was done overnight at 4°C: mouse anti-phospho-Y214-SGG (1:400; clone 5G2F12, ), rabbit anti-ITP (1:10000, ), mouse anti-PDF (1:1500, clone C7, Developmental Studies Hybridoma Bank, Iowa City, IA, United States), rabbit anti-PER (1:750, ) and chicken anti-GFP (1:750; Millipore, Upstate, Temecula, CA, United States).

    Techniques: Activity Assay, Staining, Expressing

    Constitutively active S6K increases mitochondrial aggregation and DA neuron loss in PINK1 mutants. (A) Overexpression of constitutively active S6K increased the size of swollen or aggregated mitochondria in the DA neurons of PINK1 mutants. Mitochondrially targeted GFP (mitoGFP) was expressed in the DA neurons using TH-Gal4 driver [44] to help visualize mitochondrial morphology. Brains of 7-day-old adult flies of the indicated genotypes were immunostained with anti-TH antibody (red) to label DA neuron and anti-GFP antibody (green) to label mitochondria. Images of DA neurons in the PPL1 cluster were shown. Overexpression of S6K-TE in PINK1 mutant significantly increased the size of mitochondrial aggregates in DA neurons. The scale bar represents 5 µm. (B) Comparison of mitochondrial size distribution in PINK1 mutants with or without S6K-TE overexpression. Statistical significance was determined by Student's t test (** P

    Journal: PLoS Genetics

    Article Title: Reduction of Protein Translation and Activation of Autophagy Protect against PINK1 Pathogenesis in Drosophila melanogaster

    doi: 10.1371/journal.pgen.1001237

    Figure Lengend Snippet: Constitutively active S6K increases mitochondrial aggregation and DA neuron loss in PINK1 mutants. (A) Overexpression of constitutively active S6K increased the size of swollen or aggregated mitochondria in the DA neurons of PINK1 mutants. Mitochondrially targeted GFP (mitoGFP) was expressed in the DA neurons using TH-Gal4 driver [44] to help visualize mitochondrial morphology. Brains of 7-day-old adult flies of the indicated genotypes were immunostained with anti-TH antibody (red) to label DA neuron and anti-GFP antibody (green) to label mitochondria. Images of DA neurons in the PPL1 cluster were shown. Overexpression of S6K-TE in PINK1 mutant significantly increased the size of mitochondrial aggregates in DA neurons. The scale bar represents 5 µm. (B) Comparison of mitochondrial size distribution in PINK1 mutants with or without S6K-TE overexpression. Statistical significance was determined by Student's t test (** P

    Article Snippet: Rabbit anti-Phospho-S6K (Thr398) antibody was from Cell Signaling Technology, and chicken anti-GFP antibody was purchased from Chemicon International.

    Techniques: Over Expression, Mutagenesis

    Wnt signaling acts in the ECs to regulate Tkv expression. (A–F) Germaria with compromised Wnt signaling components (Arm [A and B, BL-35004] and Lgs [C, BL-37476]) or with overexpressed TCF DN (D and E) in ECs exhibit more spectrosome-containing cells and reduced tkv k16713 reporter expression. (F) Statistical data for spectrosome-containing cells in arm i or TCF DN expressing germarium. (G) A germarium containing ECs mutant for the pygo s123 allele (generated using the MARCM system and marked with an arrowhead) exhibits ectopic spectrosome-containing cells. Genotype: hs-flp.UAS-CD8.GFP/+;tub-gal4/+;FRT82B.pygo s123 /FRT82B.tub-gal80. (H) A pygo i (NIG-11518R) germarium harbors ectopic Dad-lacZ–positive spectrosome-containing cells. (I) A pygo i (NIG-11518R-1) germarium contains more pMad-positive spectrosome-containing cells. (J) A arm i (BL-35004) germarium expresses low levels of tkv transcripts (compared with Fig. 1 H ). (K) A lgs i (BL-37476) germarium expresses low levels of Tkv in the ECs (compared with Figure 1 I ). (L) One EC mutant for pygo s123 (arrowhead) exhibits reduced Tkv expression, compared with control EC (arrow) of the same germarium. Genotype: hs-flp;FRT82B.pygo s123 /FRT82B.ubi-GFP . (M–O) Forced expression of Tkv in lgs i (BL-37476) germaria strongly suppresses the formation of ectopic spectrosome-containing cells. ***, P

    Journal: The Journal of Cell Biology

    Article Title: Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche

    doi: 10.1083/jcb.201409142

    Figure Lengend Snippet: Wnt signaling acts in the ECs to regulate Tkv expression. (A–F) Germaria with compromised Wnt signaling components (Arm [A and B, BL-35004] and Lgs [C, BL-37476]) or with overexpressed TCF DN (D and E) in ECs exhibit more spectrosome-containing cells and reduced tkv k16713 reporter expression. (F) Statistical data for spectrosome-containing cells in arm i or TCF DN expressing germarium. (G) A germarium containing ECs mutant for the pygo s123 allele (generated using the MARCM system and marked with an arrowhead) exhibits ectopic spectrosome-containing cells. Genotype: hs-flp.UAS-CD8.GFP/+;tub-gal4/+;FRT82B.pygo s123 /FRT82B.tub-gal80. (H) A pygo i (NIG-11518R) germarium harbors ectopic Dad-lacZ–positive spectrosome-containing cells. (I) A pygo i (NIG-11518R-1) germarium contains more pMad-positive spectrosome-containing cells. (J) A arm i (BL-35004) germarium expresses low levels of tkv transcripts (compared with Fig. 1 H ). (K) A lgs i (BL-37476) germarium expresses low levels of Tkv in the ECs (compared with Figure 1 I ). (L) One EC mutant for pygo s123 (arrowhead) exhibits reduced Tkv expression, compared with control EC (arrow) of the same germarium. Genotype: hs-flp;FRT82B.pygo s123 /FRT82B.ubi-GFP . (M–O) Forced expression of Tkv in lgs i (BL-37476) germaria strongly suppresses the formation of ectopic spectrosome-containing cells. ***, P

    Article Snippet: The primary antibodies used in this study are as follows: mouse monoclonal anti–α-Spectrin (3A9, 1:100; Developmental Studies Hybridoma Bank), rabbit anti–α-Spectrin (1:3,000; generated in our laboratory), rabbit anti-pMad (1:500; Cell Signaling Technology), guinea pig anti-Vasa (a gift from T. Kai), mouse anti-Bam (1:5; Developmental Studies Hybridoma Bank), chicken anti-GFP (1:5,000; Abcam), rabbit anti-Tkv (against the extracellular domain; 1:2,000; generated in this study), rabbit anti-phosphohistone H3(Ser10) (1:2,000; Cell Signaling Technology).

    Techniques: Expressing, Mutagenesis, Generated

    Dpp binding is important for the function of Tkv. (A and B) The overexpression of Tkv[EX]-GFP in the ECs of a tkv i (v105834) germarium suppresses the formation of ectopic spectrosome-containing cells. (C and D) The expression of Tkv[EX]-Flag in ECs mutant (C, arrowheads) for the tkv 8 allele partially suppresses the formation of ectopic spectrosome-containing cells. Bars, 10 µm. ***, P

    Journal: The Journal of Cell Biology

    Article Title: Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche

    doi: 10.1083/jcb.201409142

    Figure Lengend Snippet: Dpp binding is important for the function of Tkv. (A and B) The overexpression of Tkv[EX]-GFP in the ECs of a tkv i (v105834) germarium suppresses the formation of ectopic spectrosome-containing cells. (C and D) The expression of Tkv[EX]-Flag in ECs mutant (C, arrowheads) for the tkv 8 allele partially suppresses the formation of ectopic spectrosome-containing cells. Bars, 10 µm. ***, P

    Article Snippet: The primary antibodies used in this study are as follows: mouse monoclonal anti–α-Spectrin (3A9, 1:100; Developmental Studies Hybridoma Bank), rabbit anti–α-Spectrin (1:3,000; generated in our laboratory), rabbit anti-pMad (1:500; Cell Signaling Technology), guinea pig anti-Vasa (a gift from T. Kai), mouse anti-Bam (1:5; Developmental Studies Hybridoma Bank), chicken anti-GFP (1:5,000; Abcam), rabbit anti-Tkv (against the extracellular domain; 1:2,000; generated in this study), rabbit anti-phosphohistone H3(Ser10) (1:2,000; Cell Signaling Technology).

    Techniques: Binding Assay, Over Expression, Expressing, Mutagenesis

    Tkv acts in ECs to restrict germline proliferation. (A) Schematic of a Drosophila germarium. (B and C) Compared with a control ( c587ts/+ ) germarium (B), a tkv i (C; BL40937) germarium contains ectopic spectrosome-containing cells. Vasa (green) is a germ cell marker. (D) Statistical data showing the number of spectrosome-containing cells in control ( c587ts/+ ), tkv i ( c587ts;tkv i [BL-40937]), off-target construct rescue ( c587ts ; UAS-tkv [off-target]), or c587ts ; tkv i / UAS-tkv [off-target] germaria. (E–G) A germarium carrying tkv 8 mutant ECs (lack of GFP signal marked by arrowheads) exhibits more spectrosome-containing cells (E and F), which is rescued by restoring tkv expression (G). (E′) Cartoon model to illustrate the position of mutant EC clones (blue). The genotype of E is c587.UAS-flp; FRT40A.ubiGFP/FRT40A.tkv 8 . The genotype of G is c587.UAS-flp; FRT40A.ubiGFP/FRT40A.tkv 8 ;UAS-tkv . (H) In addition to the germline expression (white arrows indicate GSCs and CBs and yellow arrows indicate developing oocytes), tkv mRNA is strongly detected in the ECs (arrowheads). (I) A c 587ts;UAS-CD8GFP germarium exhibits strongly colocalized Tkv and GFP staining in ECs (arrows). Bars, 10 µm. ***, P

    Journal: The Journal of Cell Biology

    Article Title: Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche

    doi: 10.1083/jcb.201409142

    Figure Lengend Snippet: Tkv acts in ECs to restrict germline proliferation. (A) Schematic of a Drosophila germarium. (B and C) Compared with a control ( c587ts/+ ) germarium (B), a tkv i (C; BL40937) germarium contains ectopic spectrosome-containing cells. Vasa (green) is a germ cell marker. (D) Statistical data showing the number of spectrosome-containing cells in control ( c587ts/+ ), tkv i ( c587ts;tkv i [BL-40937]), off-target construct rescue ( c587ts ; UAS-tkv [off-target]), or c587ts ; tkv i / UAS-tkv [off-target] germaria. (E–G) A germarium carrying tkv 8 mutant ECs (lack of GFP signal marked by arrowheads) exhibits more spectrosome-containing cells (E and F), which is rescued by restoring tkv expression (G). (E′) Cartoon model to illustrate the position of mutant EC clones (blue). The genotype of E is c587.UAS-flp; FRT40A.ubiGFP/FRT40A.tkv 8 . The genotype of G is c587.UAS-flp; FRT40A.ubiGFP/FRT40A.tkv 8 ;UAS-tkv . (H) In addition to the germline expression (white arrows indicate GSCs and CBs and yellow arrows indicate developing oocytes), tkv mRNA is strongly detected in the ECs (arrowheads). (I) A c 587ts;UAS-CD8GFP germarium exhibits strongly colocalized Tkv and GFP staining in ECs (arrows). Bars, 10 µm. ***, P

    Article Snippet: The primary antibodies used in this study are as follows: mouse monoclonal anti–α-Spectrin (3A9, 1:100; Developmental Studies Hybridoma Bank), rabbit anti–α-Spectrin (1:3,000; generated in our laboratory), rabbit anti-pMad (1:500; Cell Signaling Technology), guinea pig anti-Vasa (a gift from T. Kai), mouse anti-Bam (1:5; Developmental Studies Hybridoma Bank), chicken anti-GFP (1:5,000; Abcam), rabbit anti-Tkv (against the extracellular domain; 1:2,000; generated in this study), rabbit anti-phosphohistone H3(Ser10) (1:2,000; Cell Signaling Technology).

    Techniques: Marker, Construct, Mutagenesis, Expressing, Staining

    Cap cell–expressed Wnts promote Tkv expression in ECs. wg (A) and wnt6 (B) transcripts are strongly detected in cap cells (indicated by strong LamC expression). Both wnt2 (C) and wnt4 (D) transcripts are detected in cap cells and ECs. (E–H) Knocking down Wnt6 (F) but not Wg (E) from cap cells results in a slight increase in spectrosome-containing cells, whereas removing both Wg and Wnt6 from cap cells leads to the formation of ectopic spectrosome-containing cells (G and H). (I) A germarium with cap cell mutants (lack of GFP signals marked by arrowheads) for a deficiency removing wg, wnt4, wnt6 , and wnt10 contains more spectrosomes. Genotype: FRT40A.ubi-GFP/FRT40A.Df(2L)Excel6017;bab1-gal4.UAS-flp . (J) Statistical data from spectrosome-containing cells in I. (K) A schematic of the genomic region used for the generation of two new transgenic reporters (P1-lacZ and P2-lacZ). (L) P1-lacZ is expressed in several ECs (arrows). (M) P2-lacZ is expressed in most ECs. (N and O) P2-lacZ (O) but not P1-lacZ (N) is down-regulated in the ECs of lgs i (BL-37476) germaria (arrows in N indicate ECs). (P) ChIP experiments in KC167 cells showing that Arm S10 is enriched at the P2 region of the tkv enhancer. Regions 1–18 cover the entire P2 region. Regions −1 to −4 outside the P2 region serve as negative controls, and N4 serves as a positive control (see Materials and methods). (Q) A luciferase assay using dissected fragments of the P2 region shows that fragment #1 responds strongly to Arm S10 overexpression. The dissected fragments (#1 to #3) are shown in the bottom panel. (R) Luciferase assay using different variants of fragment #1 with site1, site2, or site cluster3 deleted (deleted sequence is underlined); bold letters indicate the binding consensus sequence (5′-CTTTG-3′). The luminance intensity is the relative ratio of Firefly/Renilla normalized by the value of related luciferase reporter alone. Error bars represent the SEM. ***, P

    Journal: The Journal of Cell Biology

    Article Title: Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche

    doi: 10.1083/jcb.201409142

    Figure Lengend Snippet: Cap cell–expressed Wnts promote Tkv expression in ECs. wg (A) and wnt6 (B) transcripts are strongly detected in cap cells (indicated by strong LamC expression). Both wnt2 (C) and wnt4 (D) transcripts are detected in cap cells and ECs. (E–H) Knocking down Wnt6 (F) but not Wg (E) from cap cells results in a slight increase in spectrosome-containing cells, whereas removing both Wg and Wnt6 from cap cells leads to the formation of ectopic spectrosome-containing cells (G and H). (I) A germarium with cap cell mutants (lack of GFP signals marked by arrowheads) for a deficiency removing wg, wnt4, wnt6 , and wnt10 contains more spectrosomes. Genotype: FRT40A.ubi-GFP/FRT40A.Df(2L)Excel6017;bab1-gal4.UAS-flp . (J) Statistical data from spectrosome-containing cells in I. (K) A schematic of the genomic region used for the generation of two new transgenic reporters (P1-lacZ and P2-lacZ). (L) P1-lacZ is expressed in several ECs (arrows). (M) P2-lacZ is expressed in most ECs. (N and O) P2-lacZ (O) but not P1-lacZ (N) is down-regulated in the ECs of lgs i (BL-37476) germaria (arrows in N indicate ECs). (P) ChIP experiments in KC167 cells showing that Arm S10 is enriched at the P2 region of the tkv enhancer. Regions 1–18 cover the entire P2 region. Regions −1 to −4 outside the P2 region serve as negative controls, and N4 serves as a positive control (see Materials and methods). (Q) A luciferase assay using dissected fragments of the P2 region shows that fragment #1 responds strongly to Arm S10 overexpression. The dissected fragments (#1 to #3) are shown in the bottom panel. (R) Luciferase assay using different variants of fragment #1 with site1, site2, or site cluster3 deleted (deleted sequence is underlined); bold letters indicate the binding consensus sequence (5′-CTTTG-3′). The luminance intensity is the relative ratio of Firefly/Renilla normalized by the value of related luciferase reporter alone. Error bars represent the SEM. ***, P

    Article Snippet: The primary antibodies used in this study are as follows: mouse monoclonal anti–α-Spectrin (3A9, 1:100; Developmental Studies Hybridoma Bank), rabbit anti–α-Spectrin (1:3,000; generated in our laboratory), rabbit anti-pMad (1:500; Cell Signaling Technology), guinea pig anti-Vasa (a gift from T. Kai), mouse anti-Bam (1:5; Developmental Studies Hybridoma Bank), chicken anti-GFP (1:5,000; Abcam), rabbit anti-Tkv (against the extracellular domain; 1:2,000; generated in this study), rabbit anti-phosphohistone H3(Ser10) (1:2,000; Cell Signaling Technology).

    Techniques: Expressing, Transgenic Assay, Chromatin Immunoprecipitation, Positive Control, Luciferase, Over Expression, Sequencing, Binding Assay

    Simultaneous detection of vGlut2 and Gad65 in POMC neurons. A–C: POMC-EGFP was detected with a GFP antibody (A, red) after in situ hybridization for both vGlut2 using an FITC-labeled probe and TSA-Biotin detection (B, green) and Gad65 using DIG-labeled

    Journal: The Journal of comparative neurology

    Article Title: Expression of GABAergic and Glutamatergic Phenotypic Markers in Hypothalamic Proopiomelanocortin Neurons

    doi: 10.1002/cne.23127

    Figure Lengend Snippet: Simultaneous detection of vGlut2 and Gad65 in POMC neurons. A–C: POMC-EGFP was detected with a GFP antibody (A, red) after in situ hybridization for both vGlut2 using an FITC-labeled probe and TSA-Biotin detection (B, green) and Gad65 using DIG-labeled

    Article Snippet: This included the anti-FITC POD and anti-DIG AP antibodies, as well as a chicken-anti-GFP antibody at 4°C (1:2,000; Abcam, Boston, MA) to detect GFP when using the transgenic animals.

    Techniques: In Situ Hybridization, Labeling

    Fank1 and Jazf1 are specifically expressed in adult mouse airway ciliated cells and their in vitro knock-down inhibits ciliated cell differentiation. (A) Relative levels of Foxj1 , Fank1 and Jazf1 normalised to Abl1 , detected by qRT-PCR in freshly isolated adult mouse tracheal basal, ciliated and secretory cells. N =3 biological replicates. Data are mean±s.e.m. (B) Experimental set-up for MTEC ALI culture expression time-course. (C,C′) Relative levels of Fank1 , Jazf1 , Mcin , Myb , Foxj1 , Rfx3 and Cetn2 during ALI differentiation. Values normalised to Abl1 and the highest value set to 1. N =3 biological replicates. Data are mean±s.e.m. P -values are for one-way analysis of variance test for difference between groups. (D) Experimental set-up for lentiviral knock-down of Fank1 or Jazf1 in MTEC ALI cultures. (E) Representative whole-mount images of ALI cultures infected with control shRNA, Fank1 shRNA construct 2 and Jazf1 shRNA construct 4 lentiviral vectors. Green: GFP (infected cells); red: ACT (cilia); white: E-cadherin (lateral cell membranes); blue: Dapi (nuclei). Scale bar: 200 μm. (F) Quantitation of staining in E showing percentage of infected GFP + cells that are ciliated. Data are mean±s.e.m. P -values are for Student's t -test compared to control. (G) Fold change in proportion of GFP + ciliated cells compared to scrambled control following infection of lentiviral knock-down constructs. Data are mean±s.e.m. P -values are for Student's t -test compared to control. (H) Graphs to show fold change in mRNA levels of Fank1 or Jazf1 in GFP + cells following control, Fank1 or Jazf1 knock-down. Data normalised to Abl1 and the control set at 1. N =3 biological replicates. Data are mean±s.e.m.

    Journal: Biology Open

    Article Title: Fank1 and Jazf1 promote multiciliated cell differentiation in the mouse airway epithelium

    doi: 10.1242/bio.033944

    Figure Lengend Snippet: Fank1 and Jazf1 are specifically expressed in adult mouse airway ciliated cells and their in vitro knock-down inhibits ciliated cell differentiation. (A) Relative levels of Foxj1 , Fank1 and Jazf1 normalised to Abl1 , detected by qRT-PCR in freshly isolated adult mouse tracheal basal, ciliated and secretory cells. N =3 biological replicates. Data are mean±s.e.m. (B) Experimental set-up for MTEC ALI culture expression time-course. (C,C′) Relative levels of Fank1 , Jazf1 , Mcin , Myb , Foxj1 , Rfx3 and Cetn2 during ALI differentiation. Values normalised to Abl1 and the highest value set to 1. N =3 biological replicates. Data are mean±s.e.m. P -values are for one-way analysis of variance test for difference between groups. (D) Experimental set-up for lentiviral knock-down of Fank1 or Jazf1 in MTEC ALI cultures. (E) Representative whole-mount images of ALI cultures infected with control shRNA, Fank1 shRNA construct 2 and Jazf1 shRNA construct 4 lentiviral vectors. Green: GFP (infected cells); red: ACT (cilia); white: E-cadherin (lateral cell membranes); blue: Dapi (nuclei). Scale bar: 200 μm. (F) Quantitation of staining in E showing percentage of infected GFP + cells that are ciliated. Data are mean±s.e.m. P -values are for Student's t -test compared to control. (G) Fold change in proportion of GFP + ciliated cells compared to scrambled control following infection of lentiviral knock-down constructs. Data are mean±s.e.m. P -values are for Student's t -test compared to control. (H) Graphs to show fold change in mRNA levels of Fank1 or Jazf1 in GFP + cells following control, Fank1 or Jazf1 knock-down. Data normalised to Abl1 and the control set at 1. N =3 biological replicates. Data are mean±s.e.m.

    Article Snippet: Primary antibodies: mouse anti-acetylated tubulin (1:3000, Sigma-Aldrich, clone 6-11B-1); rat anti-E-cadherin (1:3000, Invitrogen, clone ECCD-2); mouse anti-Foxj1 (1:300, BD Bioscience, clone 2A5); rabbit anti-γ-tubulin (1:1000, Sigma-Aldrich, clone 310381); chick anti-GFP (1:1000, Abcam ab13970); mouse anti-p63 (1:500, Santa Cruz Biotechnology, clone 4A4); rabbit anti-Scgb1a1 (1:500; Santa Cruz Biotechnology, sc-25555).

    Techniques: In Vitro, Cell Differentiation, Quantitative RT-PCR, Isolation, Expressing, Infection, shRNA, Construct, Activated Clotting Time Assay, Quantitation Assay, Staining

    A ciliated cell-specific transcriptome for the developing mouse airways. (A) Diagram illustrating the lineage relationships between cells in the embryonic lung. Rectangles denote cell populations isolated for microarray analysis. (B) GO analysis of transcripts enriched > 3-fold in the E17.5 FOXJ1-GFP + cells compared with the E11.5 tip progenitors showed that categories associated with cilia were highly enriched compared with their frequency in the reference genome. (C) mRNA in situ hybridisation for Foxj1 , Mlf1 , Tp73 , Zcchc12 , Dlx4 , Hipk3 , Hes6 , Nr4a3 , Casz1 and Sox1 in the E17.5 stage mouse airways. Scale bars: 100 μm; 50 μm in insets.

    Journal: Biology Open

    Article Title: Fank1 and Jazf1 promote multiciliated cell differentiation in the mouse airway epithelium

    doi: 10.1242/bio.033944

    Figure Lengend Snippet: A ciliated cell-specific transcriptome for the developing mouse airways. (A) Diagram illustrating the lineage relationships between cells in the embryonic lung. Rectangles denote cell populations isolated for microarray analysis. (B) GO analysis of transcripts enriched > 3-fold in the E17.5 FOXJ1-GFP + cells compared with the E11.5 tip progenitors showed that categories associated with cilia were highly enriched compared with their frequency in the reference genome. (C) mRNA in situ hybridisation for Foxj1 , Mlf1 , Tp73 , Zcchc12 , Dlx4 , Hipk3 , Hes6 , Nr4a3 , Casz1 and Sox1 in the E17.5 stage mouse airways. Scale bars: 100 μm; 50 μm in insets.

    Article Snippet: Primary antibodies: mouse anti-acetylated tubulin (1:3000, Sigma-Aldrich, clone 6-11B-1); rat anti-E-cadherin (1:3000, Invitrogen, clone ECCD-2); mouse anti-Foxj1 (1:300, BD Bioscience, clone 2A5); rabbit anti-γ-tubulin (1:1000, Sigma-Aldrich, clone 310381); chick anti-GFP (1:1000, Abcam ab13970); mouse anti-p63 (1:500, Santa Cruz Biotechnology, clone 4A4); rabbit anti-Scgb1a1 (1:500; Santa Cruz Biotechnology, sc-25555).

    Techniques: Isolation, Microarray, In Situ, Hybridization

    Levels of ciliated cell-specific genes are decreased in the Fank1 and Jazf1 knock-downs. (A) Experimental set-up for lentiviral knock-down of Fank1 or Jazf1 in MTEC ALI cultures followed by isolation of GFP + cells at day 16 (B), or fixation for immunostaining at day 28 (C-E). (B) Graphs to show fold change in mRNA levels of Mcin , Foxj1 , Myb , Rfx3 and Spag6 following control, Fank1 or Jazf1 knock-down. Data normalised to Abl1 and the control set at 1. N =3 biological replicates. Data are mean±s.e.m. (C,D) Representative antibody staining from culture day 28. (C) Upper panels: merged images. Bottom panels: Foxj1/γ-tubulin co-localisation only. Green: GFP (lentiviral-infected cells); red: Foxj1 (ciliated cells); white: γ-tubulin (centrosomes and basal bodies). (D) Upper panels: GFP/ACT co-localisation. Lower panels: GFP/γ-tubulin co-localisation. Green: GFP (lentiviral-infected cells); red: ACT (cilia); white: γ-tubulin (centrosomes and basal bodies). All γ-tubulin + cells are also ACT + . (E) Quantification of Foxj1 staining in C showing percentage of GFP + cells that are Foxj1 + . N =3 biological replicates. Data are mean±s.e.m. P -values are for Student's t -test compared to control. Scale bars: 50 μm.

    Journal: Biology Open

    Article Title: Fank1 and Jazf1 promote multiciliated cell differentiation in the mouse airway epithelium

    doi: 10.1242/bio.033944

    Figure Lengend Snippet: Levels of ciliated cell-specific genes are decreased in the Fank1 and Jazf1 knock-downs. (A) Experimental set-up for lentiviral knock-down of Fank1 or Jazf1 in MTEC ALI cultures followed by isolation of GFP + cells at day 16 (B), or fixation for immunostaining at day 28 (C-E). (B) Graphs to show fold change in mRNA levels of Mcin , Foxj1 , Myb , Rfx3 and Spag6 following control, Fank1 or Jazf1 knock-down. Data normalised to Abl1 and the control set at 1. N =3 biological replicates. Data are mean±s.e.m. (C,D) Representative antibody staining from culture day 28. (C) Upper panels: merged images. Bottom panels: Foxj1/γ-tubulin co-localisation only. Green: GFP (lentiviral-infected cells); red: Foxj1 (ciliated cells); white: γ-tubulin (centrosomes and basal bodies). (D) Upper panels: GFP/ACT co-localisation. Lower panels: GFP/γ-tubulin co-localisation. Green: GFP (lentiviral-infected cells); red: ACT (cilia); white: γ-tubulin (centrosomes and basal bodies). All γ-tubulin + cells are also ACT + . (E) Quantification of Foxj1 staining in C showing percentage of GFP + cells that are Foxj1 + . N =3 biological replicates. Data are mean±s.e.m. P -values are for Student's t -test compared to control. Scale bars: 50 μm.

    Article Snippet: Primary antibodies: mouse anti-acetylated tubulin (1:3000, Sigma-Aldrich, clone 6-11B-1); rat anti-E-cadherin (1:3000, Invitrogen, clone ECCD-2); mouse anti-Foxj1 (1:300, BD Bioscience, clone 2A5); rabbit anti-γ-tubulin (1:1000, Sigma-Aldrich, clone 310381); chick anti-GFP (1:1000, Abcam ab13970); mouse anti-p63 (1:500, Santa Cruz Biotechnology, clone 4A4); rabbit anti-Scgb1a1 (1:500; Santa Cruz Biotechnology, sc-25555).

    Techniques: Isolation, Immunostaining, Staining, Infection, Activated Clotting Time Assay

    An ex vivo embryonic airway overexpression assay identifies Fank1 and Jazf1 as novel factors that can promote ciliated cell differentiation. (A-C) Frozen sections showing in vitro differentiation of E14.5 wild-type mouse tracheae over 7 days ex vivo . (A,B) Green=TRP63, progenitor cells; red=E-cadherin, lateral cell membranes. (C) Green=ACT, ciliated cells; red=SCGB1A1, club cells. (D) Diagram of overexpression assay. E14.5 undifferentiated tracheae are electroporated with expression constructs and cultured for 7 days. Right panel shows whole-mount image of trachea at culture day 7 stained for GFP (green) and E-cadherin (red) to illustrate the typical extent of electroporation. (E) Examples of sectioned electroporated tracheae at E14.5+7 days. Green=GFP, electroporated cells; red=ACT, cilia; blue=DAPI, nuclei. (F) Graph to show percentage of electroporated GFP + cells that co-stain with ACT in each condition tested. Data are mean±s.e.m. * P

    Journal: Biology Open

    Article Title: Fank1 and Jazf1 promote multiciliated cell differentiation in the mouse airway epithelium

    doi: 10.1242/bio.033944

    Figure Lengend Snippet: An ex vivo embryonic airway overexpression assay identifies Fank1 and Jazf1 as novel factors that can promote ciliated cell differentiation. (A-C) Frozen sections showing in vitro differentiation of E14.5 wild-type mouse tracheae over 7 days ex vivo . (A,B) Green=TRP63, progenitor cells; red=E-cadherin, lateral cell membranes. (C) Green=ACT, ciliated cells; red=SCGB1A1, club cells. (D) Diagram of overexpression assay. E14.5 undifferentiated tracheae are electroporated with expression constructs and cultured for 7 days. Right panel shows whole-mount image of trachea at culture day 7 stained for GFP (green) and E-cadherin (red) to illustrate the typical extent of electroporation. (E) Examples of sectioned electroporated tracheae at E14.5+7 days. Green=GFP, electroporated cells; red=ACT, cilia; blue=DAPI, nuclei. (F) Graph to show percentage of electroporated GFP + cells that co-stain with ACT in each condition tested. Data are mean±s.e.m. * P

    Article Snippet: Primary antibodies: mouse anti-acetylated tubulin (1:3000, Sigma-Aldrich, clone 6-11B-1); rat anti-E-cadherin (1:3000, Invitrogen, clone ECCD-2); mouse anti-Foxj1 (1:300, BD Bioscience, clone 2A5); rabbit anti-γ-tubulin (1:1000, Sigma-Aldrich, clone 310381); chick anti-GFP (1:1000, Abcam ab13970); mouse anti-p63 (1:500, Santa Cruz Biotechnology, clone 4A4); rabbit anti-Scgb1a1 (1:500; Santa Cruz Biotechnology, sc-25555).

    Techniques: Ex Vivo, Over Expression, Cell Differentiation, In Vitro, Activated Clotting Time Assay, Expressing, Construct, Cell Culture, Staining, Electroporation

    Ad5-CMV-Cre is capable of transducing AM. (A) Immunostaining for CD107b (red) and GFP (green) in a Kras LSL-G12D .mT/mG animal after Ad5-CMV-Cre treatment demonstrating co-localization of GFP in a subset of CD107b + AM. Arrows indicate GFP + /CD107b + AM. In each panel isotype controls are shown as insets, the tumor margin is highlighted in yellow, and the scale bar is 100 µm. (B) Triple immunostaining for TTF-1 (red), GFP (green), and CD107b (blue) illustrating GFP + /TTF + tumor cells and a GFP + /CD107b + AM (white arrow). (C) Dual immunostaining for CD107b (red) and Kras G12D (green) illustrating Kras G12D expression in both tumor cells and CD107b + AM. Arrows indicate Kras G12D+ /CD107 + AMs. A Kras G12D negative AM is shown at bottom left. (D) Triple immunostaining for CD107b (red), GFP (green), and PCNA (blue) showing PCNA staining in GFP + /CD107b + AM (yellow cells with blue nuclei in right panel). Five sections from 4 Ad5-CMV-Cre initiated lung tumors were quantified. A tumor infiltrating macrophage is shown in the right panel (arrow at top right); additional images of tumor infiltrating macrophages are shown as Supplemental Fig. S3.

    Journal: Oncoimmunology

    Article Title: Adenoviral vectors transduce alveolar macrophages in lung cancer models

    doi: 10.1080/2162402X.2018.1438105

    Figure Lengend Snippet: Ad5-CMV-Cre is capable of transducing AM. (A) Immunostaining for CD107b (red) and GFP (green) in a Kras LSL-G12D .mT/mG animal after Ad5-CMV-Cre treatment demonstrating co-localization of GFP in a subset of CD107b + AM. Arrows indicate GFP + /CD107b + AM. In each panel isotype controls are shown as insets, the tumor margin is highlighted in yellow, and the scale bar is 100 µm. (B) Triple immunostaining for TTF-1 (red), GFP (green), and CD107b (blue) illustrating GFP + /TTF + tumor cells and a GFP + /CD107b + AM (white arrow). (C) Dual immunostaining for CD107b (red) and Kras G12D (green) illustrating Kras G12D expression in both tumor cells and CD107b + AM. Arrows indicate Kras G12D+ /CD107 + AMs. A Kras G12D negative AM is shown at bottom left. (D) Triple immunostaining for CD107b (red), GFP (green), and PCNA (blue) showing PCNA staining in GFP + /CD107b + AM (yellow cells with blue nuclei in right panel). Five sections from 4 Ad5-CMV-Cre initiated lung tumors were quantified. A tumor infiltrating macrophage is shown in the right panel (arrow at top right); additional images of tumor infiltrating macrophages are shown as Supplemental Fig. S3.

    Article Snippet: Mice were treated with tracheal instillation of Ad5-CMV-Cre (30 µl of 109 PFU/ml) or Ad5-EA (30 μl of 5 × 108 PFU/ml) or by direct intrapulmonary injection of Ad5-CMV-Cre (2 µl of 109 PFU/ml) or Ad5-SPC-Cre (2 µl of 1010 PFU/ml)., Immunofluorescence: Immunostaining was performed as previously described with primary antibodies against GFP (1:1000; ab13970, Abcam), KrasG12D (1:50; GTX132407, GeneTex), CD107b (1:200; 550292, BD Biosciences), PCNA (1:250; ab18197, Abcam), E-Cadherin (1:200; 3195, Cell Signaling), TTF1 (1:200; ab76013, Abcam), FLAG (1:50; F4049, Sigma-Aldrich), and species-appropriate secondary antibodies: Alexa Fluor 594 (1:200; A11007 & A11012, Invitrogen), Alexa Fluor 488 (1:300; A11006, A11008 & A11039, Invitrogen), Alexa Fluor 405 (1:200; ab175652, Abcam) and Alexa Fluor 350 (1:200; A21093, Invitrogen).

    Techniques: Immunostaining, Triple Immunostaining, Expressing, Affinity Magnetic Separation, Staining

    Ad5-CMV-Cre treatment of Kras LSL-G12D .mT/mG mice causes adenocarcinoma formation. (A) H E stained tumor sections showing adenocarcinoma formation after Ad5-CMV-Cre treatment by intratracheal instillation (left) or direct injection into the left lung (right). Scale bar in all panels is 100 μm. (B) Immunostaining for E-cadherin (red) and GFP (green) in a Kras LSL-G12D .mT/mG animal following direct injection of Ad5-CMV-Cre into the left lung. Arrows indicate GFP positive, E-cadherin negative cells distinct from the tumor. In this and subsequent panels, the tumor margin is highlighted in yellow and insets show isotype controls. (C) Immunostaining for TTF-1 (red) and GFP (green). Overlay with DAPI counterstain shows co-localization of GFP and TTF-1 expression in tumor epithelial cells (GFP positive cells with pink nuclei). Arrows indicate GFP positive/TTF-1 negative cells distinct from the tumor. (D) Immunostaining for Kras G12D (red) and GFP (green) confirms Kras G12D expression in tumor cells. Arrows indicate Kras G12D positive/GFP positive cells with AM morphology at the tumor margin. No Kras G12D staining was observed in tumors driven by the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) gene fusion (Supplemental Fig. S1).

    Journal: Oncoimmunology

    Article Title: Adenoviral vectors transduce alveolar macrophages in lung cancer models

    doi: 10.1080/2162402X.2018.1438105

    Figure Lengend Snippet: Ad5-CMV-Cre treatment of Kras LSL-G12D .mT/mG mice causes adenocarcinoma formation. (A) H E stained tumor sections showing adenocarcinoma formation after Ad5-CMV-Cre treatment by intratracheal instillation (left) or direct injection into the left lung (right). Scale bar in all panels is 100 μm. (B) Immunostaining for E-cadherin (red) and GFP (green) in a Kras LSL-G12D .mT/mG animal following direct injection of Ad5-CMV-Cre into the left lung. Arrows indicate GFP positive, E-cadherin negative cells distinct from the tumor. In this and subsequent panels, the tumor margin is highlighted in yellow and insets show isotype controls. (C) Immunostaining for TTF-1 (red) and GFP (green). Overlay with DAPI counterstain shows co-localization of GFP and TTF-1 expression in tumor epithelial cells (GFP positive cells with pink nuclei). Arrows indicate GFP positive/TTF-1 negative cells distinct from the tumor. (D) Immunostaining for Kras G12D (red) and GFP (green) confirms Kras G12D expression in tumor cells. Arrows indicate Kras G12D positive/GFP positive cells with AM morphology at the tumor margin. No Kras G12D staining was observed in tumors driven by the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) gene fusion (Supplemental Fig. S1).

    Article Snippet: Mice were treated with tracheal instillation of Ad5-CMV-Cre (30 µl of 109 PFU/ml) or Ad5-EA (30 μl of 5 × 108 PFU/ml) or by direct intrapulmonary injection of Ad5-CMV-Cre (2 µl of 109 PFU/ml) or Ad5-SPC-Cre (2 µl of 1010 PFU/ml)., Immunofluorescence: Immunostaining was performed as previously described with primary antibodies against GFP (1:1000; ab13970, Abcam), KrasG12D (1:50; GTX132407, GeneTex), CD107b (1:200; 550292, BD Biosciences), PCNA (1:250; ab18197, Abcam), E-Cadherin (1:200; 3195, Cell Signaling), TTF1 (1:200; ab76013, Abcam), FLAG (1:50; F4049, Sigma-Aldrich), and species-appropriate secondary antibodies: Alexa Fluor 594 (1:200; A11007 & A11012, Invitrogen), Alexa Fluor 488 (1:300; A11006, A11008 & A11039, Invitrogen), Alexa Fluor 405 (1:200; ab175652, Abcam) and Alexa Fluor 350 (1:200; A21093, Invitrogen).

    Techniques: Mouse Assay, Staining, Injection, Immunostaining, Expressing

    Adenoviral-mediated AM transduction can be observed days after treatment. (A) GFP + AM from BAL 1 week after intratracheal Ad5-CMV-Cre treatment. mGFP and mTomato expression were quantified by fluorescent microscopy from 4 animals (350 cells per sample). Essentially all AM were mTom + while 37 ± 2% (mean ± SEM) of AM were also GFP + . Scale bar is 50 µm. (B) CD107b + AM expressing FLAG-tagged Cas9 3 days after intratracheal administration of Ad5-EA. Immunostaining was performed as described in Methods. Insets show isotype controls, the scale bar is 50 µm.

    Journal: Oncoimmunology

    Article Title: Adenoviral vectors transduce alveolar macrophages in lung cancer models

    doi: 10.1080/2162402X.2018.1438105

    Figure Lengend Snippet: Adenoviral-mediated AM transduction can be observed days after treatment. (A) GFP + AM from BAL 1 week after intratracheal Ad5-CMV-Cre treatment. mGFP and mTomato expression were quantified by fluorescent microscopy from 4 animals (350 cells per sample). Essentially all AM were mTom + while 37 ± 2% (mean ± SEM) of AM were also GFP + . Scale bar is 50 µm. (B) CD107b + AM expressing FLAG-tagged Cas9 3 days after intratracheal administration of Ad5-EA. Immunostaining was performed as described in Methods. Insets show isotype controls, the scale bar is 50 µm.

    Article Snippet: Mice were treated with tracheal instillation of Ad5-CMV-Cre (30 µl of 109 PFU/ml) or Ad5-EA (30 μl of 5 × 108 PFU/ml) or by direct intrapulmonary injection of Ad5-CMV-Cre (2 µl of 109 PFU/ml) or Ad5-SPC-Cre (2 µl of 1010 PFU/ml)., Immunofluorescence: Immunostaining was performed as previously described with primary antibodies against GFP (1:1000; ab13970, Abcam), KrasG12D (1:50; GTX132407, GeneTex), CD107b (1:200; 550292, BD Biosciences), PCNA (1:250; ab18197, Abcam), E-Cadherin (1:200; 3195, Cell Signaling), TTF1 (1:200; ab76013, Abcam), FLAG (1:50; F4049, Sigma-Aldrich), and species-appropriate secondary antibodies: Alexa Fluor 594 (1:200; A11007 & A11012, Invitrogen), Alexa Fluor 488 (1:300; A11006, A11008 & A11039, Invitrogen), Alexa Fluor 405 (1:200; ab175652, Abcam) and Alexa Fluor 350 (1:200; A21093, Invitrogen).

    Techniques: Transduction, Expressing, Microscopy, Immunostaining

    Motor neurons for pharyngeal pumping innervate the cibarial dilator musculature (CDM) ipsi- and contra-laterally. A, Simultaneous antennal nerve (AN) and cibarial dilator muscle (CDM) recordings of left and right side of the larval body of an OregonR larva. B, Under unimpaired conditions, AN motor pattern (red bars) of the left and right side is synchronous, and shows temporal correlation with the evoked postsynaptic potentials (PSPs) of the left and right CDM. C, Lesion of the right AN (1. Lesion) between the CNS and recording site results in abolishment of the corresponding AN motor pattern, whereas the PSPs on both sides of the CDM persist. Note that the diminished amplitude of the PSPs in the left CDM recording is caused by displacement of the glass electrode due to the lesion. D, Subsequent lesion of the left AN (2. Lesion) eliminated additionally the AN motor pattern on the left side and resulted in total disappearance of the PSP in left and right CDM. E , One CDM motorneuron (left side) strongly labelled in a larvae expressing 10xUAS-mCD8::GFP driven by MT11-Gal4. F , Schematic of the setup used for anterograde filling of the AN with tetramethylrhodamine-Dextran (Tmr-D). G , Tmr-D filled left AN shows axons with bilateral innervation of the CDM. H-H” , Colocalization of the Tmr-D labelled FN and one Axon of MT11-Gal4 driving 10xUAS-mCD8::GFP showing bilateral innervation of the CDM by one CDM motor neuron. I , Magnified region of the FN (marked in H” by white dotted box). Scalebars: E: 20μm,G: 50μm, H-H”: 20μm, I:10μm.

    Journal: PLoS ONE

    Article Title: Localization of Motor Neurons and Central Pattern Generators for Motor Patterns Underlying Feeding Behavior in Drosophila Larvae

    doi: 10.1371/journal.pone.0135011

    Figure Lengend Snippet: Motor neurons for pharyngeal pumping innervate the cibarial dilator musculature (CDM) ipsi- and contra-laterally. A, Simultaneous antennal nerve (AN) and cibarial dilator muscle (CDM) recordings of left and right side of the larval body of an OregonR larva. B, Under unimpaired conditions, AN motor pattern (red bars) of the left and right side is synchronous, and shows temporal correlation with the evoked postsynaptic potentials (PSPs) of the left and right CDM. C, Lesion of the right AN (1. Lesion) between the CNS and recording site results in abolishment of the corresponding AN motor pattern, whereas the PSPs on both sides of the CDM persist. Note that the diminished amplitude of the PSPs in the left CDM recording is caused by displacement of the glass electrode due to the lesion. D, Subsequent lesion of the left AN (2. Lesion) eliminated additionally the AN motor pattern on the left side and resulted in total disappearance of the PSP in left and right CDM. E , One CDM motorneuron (left side) strongly labelled in a larvae expressing 10xUAS-mCD8::GFP driven by MT11-Gal4. F , Schematic of the setup used for anterograde filling of the AN with tetramethylrhodamine-Dextran (Tmr-D). G , Tmr-D filled left AN shows axons with bilateral innervation of the CDM. H-H” , Colocalization of the Tmr-D labelled FN and one Axon of MT11-Gal4 driving 10xUAS-mCD8::GFP showing bilateral innervation of the CDM by one CDM motor neuron. I , Magnified region of the FN (marked in H” by white dotted box). Scalebars: E: 20μm,G: 50μm, H-H”: 20μm, I:10μm.

    Article Snippet: The sample of remaining CNS, CPS and nerves of interest was stained using anti-chicken-GFP (1:500, Abcam plc) and anti-mouse-mRFP (1:500) as primary antibodies.

    Techniques: Expressing

    All fin mesenchyme cells derive from paraxial mesoderm. ( A ) Confocal image of the tail region of a 48-hpf ntla:gal4; uas:kaede embryo. ( B-B ′) Immunofluorescent staining of 48-hpf ntla:gal4; uas:kaede ; ET37 triple transgenic embryo showing total overlap (B′) of Kaede signal (red, B,B′) and eGFP (green, B′,B′) in the fin. ( C-E ′) Fluorescent images alone (C,D,E) and superimposed on Nomarski images (C′,D′,E′) of the trunk/tail of 48-hpf embryos. The myotome (C,C′), sclerotome (D,D′) and dermomyotome (E,E′) are labelled by actc1b:Gal4 i269 ; uas:kaede (C,C′), Ola-Twist:Gal4; uas:kaede (D,D′) and TgBAC(pax3a:EGFP) i150 (E,E′) transgenics, respectively. ( F Movie 4) of the tail region of a TgBAC(pax3a:EGFP) i150 embryo at 24 hpf (left panel) with subsequent time points at given intervals (in minutes) in the panels to the right. Two fin mesenchyme cells can be tracked (arrows) from the dermomyotome into the fins. Note that eGFP expression is higher in neural crest and dorsal neural tube than in dermomyotome.

    Journal: Development (Cambridge, England)

    Article Title: An exclusively mesodermal origin of fin mesenchyme demonstrates that zebrafish trunk neural crest does not generate ectomesenchyme

    doi: 10.1242/dev.093534

    Figure Lengend Snippet: All fin mesenchyme cells derive from paraxial mesoderm. ( A ) Confocal image of the tail region of a 48-hpf ntla:gal4; uas:kaede embryo. ( B-B ′) Immunofluorescent staining of 48-hpf ntla:gal4; uas:kaede ; ET37 triple transgenic embryo showing total overlap (B′) of Kaede signal (red, B,B′) and eGFP (green, B′,B′) in the fin. ( C-E ′) Fluorescent images alone (C,D,E) and superimposed on Nomarski images (C′,D′,E′) of the trunk/tail of 48-hpf embryos. The myotome (C,C′), sclerotome (D,D′) and dermomyotome (E,E′) are labelled by actc1b:Gal4 i269 ; uas:kaede (C,C′), Ola-Twist:Gal4; uas:kaede (D,D′) and TgBAC(pax3a:EGFP) i150 (E,E′) transgenics, respectively. ( F Movie 4) of the tail region of a TgBAC(pax3a:EGFP) i150 embryo at 24 hpf (left panel) with subsequent time points at given intervals (in minutes) in the panels to the right. Two fin mesenchyme cells can be tracked (arrows) from the dermomyotome into the fins. Note that eGFP expression is higher in neural crest and dorsal neural tube than in dermomyotome.

    Article Snippet: Primary antibodies were: rat anti-eGFP (1:250; 04404-26, Nacalai Tesque), chicken anti-eGFP (1:500; ab13970, Abcam), rabbit anti-Kaede (1:250; PM012, MBL International), rabbit anti-DsRed/mCherry (1:250; 632496, BD Biosciences), mouse anti-Pax3/7 [1:200; DP312 ( )] and mouse zns-5 (1:200; Zebrafish International Resource Center).

    Techniques: Staining, Transgenic Assay, Expressing

    Neural crest cells do not contribute to fin mesenchyme. ( A-B ′) Lateral trunk (A-A′) and tail region (B,B′) of 48-hpf sox10:gal4; uas:kaede transgenic embryos. Kaede protein fluorescence (green) is observed in melanophores (A-A′), nascent dorsal root ganglia and spinal nerves (A), as well as in the fin (B,B′). ( C-C ′) Immunofluorescent labelling of Kaede (red, C), eGFP (green, C′) and merged image (C′) of the fin of a sox10:gal4; uas:kaede ; ET37 transgenic embryo. ( D-G ) Overviews (D,E) and Nomarski images (F,G) of 48-hpf wild-type (WT) (D,F) and mos -/- ; mob -/- (E,G) embryos showing loss of pigment but presence of a fully formed medial fin (E) with fin mesenchyme cells (G) in the double mutant. Red brackets indicate the region used for quantifying fin mesenchyme cells in H. ( H ) Quantification of fin mesenchyme cells in 12 WT and 12 mos -/- ; mob -/- embryos at 48 hpf. No significant differences (n.s.) were observed (two-tailed Student’s t -test). Bars indicate the mean.

    Journal: Development (Cambridge, England)

    Article Title: An exclusively mesodermal origin of fin mesenchyme demonstrates that zebrafish trunk neural crest does not generate ectomesenchyme

    doi: 10.1242/dev.093534

    Figure Lengend Snippet: Neural crest cells do not contribute to fin mesenchyme. ( A-B ′) Lateral trunk (A-A′) and tail region (B,B′) of 48-hpf sox10:gal4; uas:kaede transgenic embryos. Kaede protein fluorescence (green) is observed in melanophores (A-A′), nascent dorsal root ganglia and spinal nerves (A), as well as in the fin (B,B′). ( C-C ′) Immunofluorescent labelling of Kaede (red, C), eGFP (green, C′) and merged image (C′) of the fin of a sox10:gal4; uas:kaede ; ET37 transgenic embryo. ( D-G ) Overviews (D,E) and Nomarski images (F,G) of 48-hpf wild-type (WT) (D,F) and mos -/- ; mob -/- (E,G) embryos showing loss of pigment but presence of a fully formed medial fin (E) with fin mesenchyme cells (G) in the double mutant. Red brackets indicate the region used for quantifying fin mesenchyme cells in H. ( H ) Quantification of fin mesenchyme cells in 12 WT and 12 mos -/- ; mob -/- embryos at 48 hpf. No significant differences (n.s.) were observed (two-tailed Student’s t -test). Bars indicate the mean.

    Article Snippet: Primary antibodies were: rat anti-eGFP (1:250; 04404-26, Nacalai Tesque), chicken anti-eGFP (1:500; ab13970, Abcam), rabbit anti-Kaede (1:250; PM012, MBL International), rabbit anti-DsRed/mCherry (1:250; 632496, BD Biosciences), mouse anti-Pax3/7 [1:200; DP312 ( )] and mouse zns-5 (1:200; Zebrafish International Resource Center).

    Techniques: Transgenic Assay, Fluorescence, Mutagenesis, Two Tailed Test

    Expression of the FRET glucose sensor in neocortical slices. Representative single plane confocal image of a slice expressing the FLII12Pglu-700 μδ6 biosensor following sindbis viral transduction. Cells expressing the FRET glucose sensor are identified by their GFP fluorescence ( green ). Pyramidal cells are immunolabeled for the Satb2 transcription factor ( red ). Dashed line represents layer I and II border. Note that most layer II and III transduced cells are stab2 positive.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Supragranular Pyramidal Cells Exhibit Early Metabolic Alterations in the 3xTg-AD Mouse Model of Alzheimer’s Disease

    doi: 10.3389/fncel.2018.00216

    Figure Lengend Snippet: Expression of the FRET glucose sensor in neocortical slices. Representative single plane confocal image of a slice expressing the FLII12Pglu-700 μδ6 biosensor following sindbis viral transduction. Cells expressing the FRET glucose sensor are identified by their GFP fluorescence ( green ). Pyramidal cells are immunolabeled for the Satb2 transcription factor ( red ). Dashed line represents layer I and II border. Note that most layer II and III transduced cells are stab2 positive.

    Article Snippet: Then, slices were rinsed with phosphate-buffer saline (PBS), permeabilized with PBS/gelatin 0.2%/Triton 0.25%, and incubated overnight at 4°C with rabbit anti-Satb2 (1:1000, ab34735, Abcam; ) and chicken anti-GFP (1:1000, GFP-1020, Aves Labs; ).

    Techniques: Expressing, Transduction, Fluorescence, Immunolabeling

    DMRT5 and EMX2 bind a Gsx2 ventral-specific telencephalon enhancer. A , UCSC genome browser view of the Gsx2 locus with the location of the two cloned fragments tested in transgenic embryos. The identified putative DMRT3/5 and EMX2 BSs are shown. B , Left, A lateral view and a coronal section of the head of a E12.5 Gsx2 1.8 kb enhancer-LacZ reporter transgenic embryo (Construct A). Scale bars: lateral view, 500 μm; coronal section, 200 μm. Dashed line indicates the level of the section. Right, A coronal section of the brain of a Gsx2 1.3 kb enhancer-GFP reporter transgenic embryo (Construct B) processed by DAB immunostaining for GFP and a high-magnification view of the LGE region processed by IF for both GSX2 (red) and GFP (green). C , EMSA showing in vitro binding of cellular extracts containing DMRT3, DMRT5, and EMX2 or control extracts to BS3 of the Gsx2 enhancer. DMRT3/5 and EMX2 complex formation is competed by WT enhancer oligonucleotides but not by oligonucleotides containing mutations in the DMRT and EMX2 BSs. Arrowhead indicates a nonspecific band.

    Journal: The Journal of Neuroscience

    Article Title: DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium–Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors

    doi: 10.1523/JNEUROSCI.0375-18.2018

    Figure Lengend Snippet: DMRT5 and EMX2 bind a Gsx2 ventral-specific telencephalon enhancer. A , UCSC genome browser view of the Gsx2 locus with the location of the two cloned fragments tested in transgenic embryos. The identified putative DMRT3/5 and EMX2 BSs are shown. B , Left, A lateral view and a coronal section of the head of a E12.5 Gsx2 1.8 kb enhancer-LacZ reporter transgenic embryo (Construct A). Scale bars: lateral view, 500 μm; coronal section, 200 μm. Dashed line indicates the level of the section. Right, A coronal section of the brain of a Gsx2 1.3 kb enhancer-GFP reporter transgenic embryo (Construct B) processed by DAB immunostaining for GFP and a high-magnification view of the LGE region processed by IF for both GSX2 (red) and GFP (green). C , EMSA showing in vitro binding of cellular extracts containing DMRT3, DMRT5, and EMX2 or control extracts to BS3 of the Gsx2 enhancer. DMRT3/5 and EMX2 complex formation is competed by WT enhancer oligonucleotides but not by oligonucleotides containing mutations in the DMRT and EMX2 BSs. Arrowhead indicates a nonspecific band.

    Article Snippet: Expression of the GFP transgene was examined by DAB staining of a E12.5 brain sections with chicken anti-GFP antibodies (1:1000, Aves Labs), followed by biotinylated donkey anti-chicken IgY (1:200, Jackson ImmunoResearch Laboratories) and ABC solution (Vector Laboratories).

    Techniques: Clone Assay, Transgenic Assay, Construct, Immunostaining, In Vitro, Binding Assay

    DMRT5 forms a boundary with GSX2 at the PSB, and the overexpression of Gsx2 represses Dmrt5 expression. A , Coronal sections of the head of E14.5 Gsx2 GFP /+ knock-in embryos processed by IF with DMRT5 and GFP antibodies showing that, at the PSB, cells expressing DMRT5 do not express GFP, hence, GSX2. Boxed area is shown at a high magnification on the left. Oe, Olfactory epithelium. Scale bar, 200 μm. B–E , Coronal sections through the telencephalon of E12.5 WT or Foxg1 t TA /+ ; tet-O-Gsx2-IRES-EGFP double-transgenic embryos immunostained with the indicated antibodies. In these transgenic embryos, Gsx2 is misexpressed throughout the embryonic telencephalon and Dmrt5 is reduced. B , E , Arrows point to the ventral limit of high Dmrt5 expression.

    Journal: The Journal of Neuroscience

    Article Title: DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium–Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors

    doi: 10.1523/JNEUROSCI.0375-18.2018

    Figure Lengend Snippet: DMRT5 forms a boundary with GSX2 at the PSB, and the overexpression of Gsx2 represses Dmrt5 expression. A , Coronal sections of the head of E14.5 Gsx2 GFP /+ knock-in embryos processed by IF with DMRT5 and GFP antibodies showing that, at the PSB, cells expressing DMRT5 do not express GFP, hence, GSX2. Boxed area is shown at a high magnification on the left. Oe, Olfactory epithelium. Scale bar, 200 μm. B–E , Coronal sections through the telencephalon of E12.5 WT or Foxg1 t TA /+ ; tet-O-Gsx2-IRES-EGFP double-transgenic embryos immunostained with the indicated antibodies. In these transgenic embryos, Gsx2 is misexpressed throughout the embryonic telencephalon and Dmrt5 is reduced. B , E , Arrows point to the ventral limit of high Dmrt5 expression.

    Article Snippet: Expression of the GFP transgene was examined by DAB staining of a E12.5 brain sections with chicken anti-GFP antibodies (1:1000, Aves Labs), followed by biotinylated donkey anti-chicken IgY (1:200, Jackson ImmunoResearch Laboratories) and ABC solution (Vector Laboratories).

    Techniques: Over Expression, Expressing, Knock-In, Transgenic Assay

    Cocaine-induced GFP expression in nucleus accumbens. (A) Representative images of cocaine-induced GFP expression in nucleus accumbens. White arrows indicate GFP-expressing cells as measured by native GFP fluorescence in c-fos-GFP transgenic mice. (B) Double-labeling immunofluorescence histochemistry of GFP (green) and the medium spiny neuron marker DARPP-32 (red) from a cocaine-sensitized mouse. White arrows indicate cells positive for both GFP and DARPP-32. (C) Co-labeling of GFP (green) and Fos (red) using immunofluorescence histochemistry from a cocaine-sensitized mouse. White arrows indicate cells positive for both GFP and Fos.

    Journal: Nature neuroscience

    Article Title: Silent synapses in selectively activated nucleus accumbens neurons following cocaine-sensitization

    doi: 10.1038/nn.3232

    Figure Lengend Snippet: Cocaine-induced GFP expression in nucleus accumbens. (A) Representative images of cocaine-induced GFP expression in nucleus accumbens. White arrows indicate GFP-expressing cells as measured by native GFP fluorescence in c-fos-GFP transgenic mice. (B) Double-labeling immunofluorescence histochemistry of GFP (green) and the medium spiny neuron marker DARPP-32 (red) from a cocaine-sensitized mouse. White arrows indicate cells positive for both GFP and DARPP-32. (C) Co-labeling of GFP (green) and Fos (red) using immunofluorescence histochemistry from a cocaine-sensitized mouse. White arrows indicate cells positive for both GFP and Fos.

    Article Snippet: We assessed co-expression of GFP and Fos in accumbens sections from 3 mice from the Repeated cocaine group using rabbit anti-c-Fos primary antibody (1:500 dilution; #c-Fos sc-52, Santa Cruz Biotechnology) and chicken anti-GFP primary antibody (1:500 dilution; #GFP-1020, Aves Labs) primary antibodies and Streptavidin Alexa 350 (1:200 dilution; #S11249, Invitrogen) and biotinylated donkey anti-chicken IgG (1:200; #703-065-155, Jackson Immunoresearch), and Alexa 568 conjugated goat anti-rabbit IgG secondary antibodies (#A11011, Invitrogen).

    Techniques: Expressing, Fluorescence, Transgenic Assay, Mouse Assay, Labeling, Immunofluorescence, Marker

    Characterization of In Vivo- Reprogrammed Cell Clusters in the GAs of Nanog-GFP and Pax3-GFP Mice (A) Nanog-GFP and Pax3-GFP transgenics were administered, 50 μg pOKS and 50 μg pM in 50 μL 0.9% saline, into the GA and dissected 2 days p.i. for histological analysis. (B) Clusters of reprogrammed cells were identified by H E and the green fluorescence resulting from either Nanog or Pax3 upregulation (100×; scale bars represent 50 μm). Bright-field and fluorescence images show the same region within the tissue. *p

    Journal: Molecular Therapy

    Article Title: Non-viral, Tumor-free Induction of Transient Cell Reprogramming in Mouse Skeletal Muscle to Enhance Tissue Regeneration

    doi: 10.1016/j.ymthe.2018.10.014

    Figure Lengend Snippet: Characterization of In Vivo- Reprogrammed Cell Clusters in the GAs of Nanog-GFP and Pax3-GFP Mice (A) Nanog-GFP and Pax3-GFP transgenics were administered, 50 μg pOKS and 50 μg pM in 50 μL 0.9% saline, into the GA and dissected 2 days p.i. for histological analysis. (B) Clusters of reprogrammed cells were identified by H E and the green fluorescence resulting from either Nanog or Pax3 upregulation (100×; scale bars represent 50 μm). Bright-field and fluorescence images show the same region within the tissue. *p

    Article Snippet: Sections were then incubated for 1 hr in blocking buffer (5% goat serum-0.1% Triton in PBS [pH 7.3]) at RT, followed by two washing steps with PBS (1% BSA- 0.1% Triton [pH 7.3]) and overnight incubation at +4°C with the following primary antibodies: rabbit polyclonal antibody (pAb) anti-NANOG (ab80892, 1/200, Abcam, UK) and chicken pAb anti-GFP (ab13970, 1/1,000, Abcam, UK).

    Techniques: In Vivo, Mouse Assay, Fluorescence

    Changes in Gene Expression after i.m. Administration of Reprogramming pDNA in Nanog-GFP Mice (A) Nanog-GFP transgenics were i.m. administered, 50 μg pOKS and 50 μg pM in 50 μL 0.9% saline or 50 μL saline alone, into the GA. (B–F) At 2, 4, and 8 days p.i. GA tissues were dissected, and real-time qRT-PCR was performed to determine the relative gene expression of (B) reprogramming factors, (C) endogenous pluripotency markers, (D) genes involved in myogenesis, (E) satellite cell markers, and (F) pericyte markers. Relative expression was normalized to day 2 values ( Oct3/4 and GFP mRNAs) or to saline-injected controls (other genes). *p

    Journal: Molecular Therapy

    Article Title: Non-viral, Tumor-free Induction of Transient Cell Reprogramming in Mouse Skeletal Muscle to Enhance Tissue Regeneration

    doi: 10.1016/j.ymthe.2018.10.014

    Figure Lengend Snippet: Changes in Gene Expression after i.m. Administration of Reprogramming pDNA in Nanog-GFP Mice (A) Nanog-GFP transgenics were i.m. administered, 50 μg pOKS and 50 μg pM in 50 μL 0.9% saline or 50 μL saline alone, into the GA. (B–F) At 2, 4, and 8 days p.i. GA tissues were dissected, and real-time qRT-PCR was performed to determine the relative gene expression of (B) reprogramming factors, (C) endogenous pluripotency markers, (D) genes involved in myogenesis, (E) satellite cell markers, and (F) pericyte markers. Relative expression was normalized to day 2 values ( Oct3/4 and GFP mRNAs) or to saline-injected controls (other genes). *p

    Article Snippet: Sections were then incubated for 1 hr in blocking buffer (5% goat serum-0.1% Triton in PBS [pH 7.3]) at RT, followed by two washing steps with PBS (1% BSA- 0.1% Triton [pH 7.3]) and overnight incubation at +4°C with the following primary antibodies: rabbit polyclonal antibody (pAb) anti-NANOG (ab80892, 1/200, Abcam, UK) and chicken pAb anti-GFP (ab13970, 1/1,000, Abcam, UK).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, Injection

    Lsd1 is required for development and maintenance of cold-induced beige adipocytes. ( A ) H E staining and ( B ) immunofluorescence detection using anti-GFP and anti-Plin1 antibodies on representative sections of ingWAT of control (Ctrl) and Lsd1 iKO-GFP mice in the absence or presence of Tam treatment. Time scale indicates age of the mice and start and end of Tam and cold treatment. Dagger indicates the time point at which mice were killed. (Scale bars: 200 µm.) ( C ) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT extracts from control and Lsd1 iKO-GFP mice treated as described in A (mean ± SEM; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Lsd1 prevents age-programed loss of beige adipocytes

    doi: 10.1073/pnas.1702641114

    Figure Lengend Snippet: Lsd1 is required for development and maintenance of cold-induced beige adipocytes. ( A ) H E staining and ( B ) immunofluorescence detection using anti-GFP and anti-Plin1 antibodies on representative sections of ingWAT of control (Ctrl) and Lsd1 iKO-GFP mice in the absence or presence of Tam treatment. Time scale indicates age of the mice and start and end of Tam and cold treatment. Dagger indicates the time point at which mice were killed. (Scale bars: 200 µm.) ( C ) qRT-PCR analysis showing relative mRNA levels of indicated genes in ingWAT extracts from control and Lsd1 iKO-GFP mice treated as described in A (mean ± SEM; * P

    Article Snippet: H & E staining, immunohistochemistry, and immunofluorescence analyses were performed as described previously ( , ) with anti-Lsd1 (R.S. laboratory [ ], 1/1,000), anti-Ucp1 (ab10983, 1/500; Abcam), or anti-GFP (ab13970, 1/500; Abcam) antibodies.

    Techniques: Staining, Immunofluorescence, Mouse Assay, Quantitative RT-PCR

    Localization of SV2C to plasma membranes mediated binding of BoNT/D-HCR to the cell surface in both neurons and HEK293FT cells. A) SV2A/B KO neurons were transfected with SV2A, B, or C. Transfected cells were marked by co-expressed GFP. Cells were exposed to BoNT/D-HCR (80 nM, 5 min in high K + buffer), washed, fixed, and permeabilized for immunostaining. Binding of BoNT/D-HCR was observed as puncta along processes in neurons transfected with SV2A (upper panel) or SV2B (middle panel). Binding of BoNT/D-HCR to SV2C-transfected neurons appears to be continuous along neuronal processes (lower panel). B) SV2A/B KO neurons were transfected with SV2C. Cells were exposed to BoNT/A (20 nM, upper panel) or BoNT/D-HCR (80 nM, lower panel) for 10 min in cold media (incubation on ice). Cells were then washed in cold media and fixed. Immunostaining was first performed without permeabilizing cells to detect the surface binding of BoNT/A or BoNT/D-HCR. Cells were subsequently permeabilized and immunostaining was further carried out to label neuronal processes using an anti-GFP antibody and presynaptic terminals using antibodies for Syb (upper panel) or synapsin (lower panel). Transfection of SV2C resulted in the binding of BoNT/A (upper panel) and BoNT/D-HCR (lower panel) to neuronal surface at both presynaptic terminals (co-localized with Syb (green) or synapsin (green)) as well as regions outside the synapse. C) Cultured rat hippocampal neurons were transfected with either Syt I (upper panel) or SV2C (lower panel). Neurons were exposed to BoNT/B (20 nM) and an antibody that recognizes the luminal domain of Syt I (Syt I N -Ab, 1∶200) for 10 min in cold media, washed, and fixed. Immunostaining was first performed without permeabilizing cells to detect the surface binding of BoNT/B and Syt I N -Ab. Cells were subsequently permeabilized and immunostaining was carried out using an anti-GFP antibody. Transfection of Syt I, but not SV2C, resulted in the binding of both BoNT/B and Syt I N -Ab to neuronal surfaces. D) Experiments were carried out as described in panel C, except that neurons were exposed to BoNT/D-HCR (80 nM). Expression of SV2C, but not Syt I, mediated the binding of BoNT/D-HCR to neuronal surfaces. E) HEK293FT cells were transfected with SV2C and exposed to BoNT/D-HCR (80 nM, 30 min in media at 37°C). Immunostaining was first carried out without permeabilizing cells to detect BoNT/D-HCR. Cells were subsequently permeabilized and immunostaining was carried out using a polyclonal anti-SV2C antibody. Expression of SV2C mediated the binding of BoNT/D-HCR to the surface of HEK293FT cells.

    Journal: PLoS Pathogens

    Article Title: Botulinum Neurotoxin D Uses Synaptic Vesicle Protein SV2 and Gangliosides as Receptors

    doi: 10.1371/journal.ppat.1002008

    Figure Lengend Snippet: Localization of SV2C to plasma membranes mediated binding of BoNT/D-HCR to the cell surface in both neurons and HEK293FT cells. A) SV2A/B KO neurons were transfected with SV2A, B, or C. Transfected cells were marked by co-expressed GFP. Cells were exposed to BoNT/D-HCR (80 nM, 5 min in high K + buffer), washed, fixed, and permeabilized for immunostaining. Binding of BoNT/D-HCR was observed as puncta along processes in neurons transfected with SV2A (upper panel) or SV2B (middle panel). Binding of BoNT/D-HCR to SV2C-transfected neurons appears to be continuous along neuronal processes (lower panel). B) SV2A/B KO neurons were transfected with SV2C. Cells were exposed to BoNT/A (20 nM, upper panel) or BoNT/D-HCR (80 nM, lower panel) for 10 min in cold media (incubation on ice). Cells were then washed in cold media and fixed. Immunostaining was first performed without permeabilizing cells to detect the surface binding of BoNT/A or BoNT/D-HCR. Cells were subsequently permeabilized and immunostaining was further carried out to label neuronal processes using an anti-GFP antibody and presynaptic terminals using antibodies for Syb (upper panel) or synapsin (lower panel). Transfection of SV2C resulted in the binding of BoNT/A (upper panel) and BoNT/D-HCR (lower panel) to neuronal surface at both presynaptic terminals (co-localized with Syb (green) or synapsin (green)) as well as regions outside the synapse. C) Cultured rat hippocampal neurons were transfected with either Syt I (upper panel) or SV2C (lower panel). Neurons were exposed to BoNT/B (20 nM) and an antibody that recognizes the luminal domain of Syt I (Syt I N -Ab, 1∶200) for 10 min in cold media, washed, and fixed. Immunostaining was first performed without permeabilizing cells to detect the surface binding of BoNT/B and Syt I N -Ab. Cells were subsequently permeabilized and immunostaining was carried out using an anti-GFP antibody. Transfection of Syt I, but not SV2C, resulted in the binding of both BoNT/B and Syt I N -Ab to neuronal surfaces. D) Experiments were carried out as described in panel C, except that neurons were exposed to BoNT/D-HCR (80 nM). Expression of SV2C, but not Syt I, mediated the binding of BoNT/D-HCR to neuronal surfaces. E) HEK293FT cells were transfected with SV2C and exposed to BoNT/D-HCR (80 nM, 30 min in media at 37°C). Immunostaining was first carried out without permeabilizing cells to detect BoNT/D-HCR. Cells were subsequently permeabilized and immunostaining was carried out using a polyclonal anti-SV2C antibody. Expression of SV2C mediated the binding of BoNT/D-HCR to the surface of HEK293FT cells.

    Article Snippet: The following antibodies were purchased from indicated vendors: mouse monoclonal anti-HA (16B12, Covance); rabbit polyclonal anti-synapsin and guinea pig anti-vesicular glutamate transporter I (vGlut-I, Millipore) ; chicken polyclonal anti-GFP and mouse monoclonal anti-actin (Abcam).

    Techniques: Binding Assay, Transfection, Immunostaining, Incubation, Cell Culture, Expressing

    BoNT/D utilizes a SV2-binding mechanism distinct from BoNT/A and E. A) Schematic drawing of SV2. Filled circles indicate conserved residues in all SV2 isoforms; gray circles are residues conserved in two SV2 isoforms; and open circles represent non-conserved residues. Three glycosylation sites within the L4 domain are also indicated. B) SV2A/B KO neurons were transfected with a chimeric receptor containing the SV2C-L4 at the N-terminus of the transmembrane and cytosolic domain of LDL receptor (SV2C-L4-LDLR, [51] ). Cells were exposed to either BoNT/A (20 nM) or BoNT/D-HCR (80 nM) for 20 min in culture media, washed and fixed for immunostaining analysis. Transfected cells were marked by co-expressed GFP. SV2C-L4-LDLR mediated the binding of BoNT/A, but failed to mediate the binding of BoNT/D-HCR. C) Chimeric protein Syg-SV2A-L4 was constructed by inserting SV2A-L4 into the second luminal domain of Syg. Syg and Syg-SV2A-L4 were expressed in SV2A/B KO neurons via lentiviral infection. Cells were exposed to BoNT/A (10 nM) and BoNT/D (100 pM). Actin serves as a loading control. Expression of Syg and Syg-SV2A-L4 was confirmed using a polyclonal anti-Syg antibody. Cleavage of SNAP-25 by BoNT/A yielded a smaller fragment that is indicated by an asterisk. Syg-SV2A-L4 mediated the entry of BoNT/A as indicated by the cleavage of SNAP-25, but failed to mediate the entry of BoNT/D as shown by the lack of Syb cleavage. D) Experiments were carried out as described in panel C, except that neurons were exposed to BoNT/E (200 pM) and BoNT/D (100 pM). Cleavage of SNAP-25 by BoNT/E yielded a smaller fragment that is indicated by an asterisk. Syx was used as a loading control. Syg-SV2A-L4 mediated the functional entry of BoNT/E, but not BoNT/D. E) Three glycosylation sites within the SV2A-L4 domain were abolished by site-directed mutagenesis, respectively. These mutants were expressed in SV2 A/B KO neurons via lentiviral infection. Neurons were exposed to BoNT/D (100 pM) and BoNT/E (200 pM). Syp serves as a loading control. Mutation N573Q abolished the entry of BoNT/E. Entry of BoNT/D was not affected by abolishing any one of the three glycosylation sites. F) SV2A/B KO neurons were infected with lentiviruses that express either WT SV2A or SV2A N573Q mutant. Cells were exposed to BoNT/A and BoNT/D at indicated concentrations (5 min in high K + buffer, 12 hrs incubation in media afterwards). Syt I serves as a loading control. Mutation N573Q reduced the cleavage of SNAP-25 by BoNT/A at low toxin concentrations (1 and 3 nM). Cleavage of Syb by BoNT/D is similar at all toxin concentrations between neurons expressing WT SV2A and N573Q mutant. G) SV2A mutants harboring disrupted luminal domain 1 (SV2A-ΔL1) or L3 (SV2A-ΔL3) were expressed in SV2A/B KO neurons via lentiviral infection. Neurons were exposed to BoNT/E (100 pM) and BoNT/D (100 pM). Disrupting L1 or L3 domain did not reduce the functional entry of either BoNT/E or BoNT/D.

    Journal: PLoS Pathogens

    Article Title: Botulinum Neurotoxin D Uses Synaptic Vesicle Protein SV2 and Gangliosides as Receptors

    doi: 10.1371/journal.ppat.1002008

    Figure Lengend Snippet: BoNT/D utilizes a SV2-binding mechanism distinct from BoNT/A and E. A) Schematic drawing of SV2. Filled circles indicate conserved residues in all SV2 isoforms; gray circles are residues conserved in two SV2 isoforms; and open circles represent non-conserved residues. Three glycosylation sites within the L4 domain are also indicated. B) SV2A/B KO neurons were transfected with a chimeric receptor containing the SV2C-L4 at the N-terminus of the transmembrane and cytosolic domain of LDL receptor (SV2C-L4-LDLR, [51] ). Cells were exposed to either BoNT/A (20 nM) or BoNT/D-HCR (80 nM) for 20 min in culture media, washed and fixed for immunostaining analysis. Transfected cells were marked by co-expressed GFP. SV2C-L4-LDLR mediated the binding of BoNT/A, but failed to mediate the binding of BoNT/D-HCR. C) Chimeric protein Syg-SV2A-L4 was constructed by inserting SV2A-L4 into the second luminal domain of Syg. Syg and Syg-SV2A-L4 were expressed in SV2A/B KO neurons via lentiviral infection. Cells were exposed to BoNT/A (10 nM) and BoNT/D (100 pM). Actin serves as a loading control. Expression of Syg and Syg-SV2A-L4 was confirmed using a polyclonal anti-Syg antibody. Cleavage of SNAP-25 by BoNT/A yielded a smaller fragment that is indicated by an asterisk. Syg-SV2A-L4 mediated the entry of BoNT/A as indicated by the cleavage of SNAP-25, but failed to mediate the entry of BoNT/D as shown by the lack of Syb cleavage. D) Experiments were carried out as described in panel C, except that neurons were exposed to BoNT/E (200 pM) and BoNT/D (100 pM). Cleavage of SNAP-25 by BoNT/E yielded a smaller fragment that is indicated by an asterisk. Syx was used as a loading control. Syg-SV2A-L4 mediated the functional entry of BoNT/E, but not BoNT/D. E) Three glycosylation sites within the SV2A-L4 domain were abolished by site-directed mutagenesis, respectively. These mutants were expressed in SV2 A/B KO neurons via lentiviral infection. Neurons were exposed to BoNT/D (100 pM) and BoNT/E (200 pM). Syp serves as a loading control. Mutation N573Q abolished the entry of BoNT/E. Entry of BoNT/D was not affected by abolishing any one of the three glycosylation sites. F) SV2A/B KO neurons were infected with lentiviruses that express either WT SV2A or SV2A N573Q mutant. Cells were exposed to BoNT/A and BoNT/D at indicated concentrations (5 min in high K + buffer, 12 hrs incubation in media afterwards). Syt I serves as a loading control. Mutation N573Q reduced the cleavage of SNAP-25 by BoNT/A at low toxin concentrations (1 and 3 nM). Cleavage of Syb by BoNT/D is similar at all toxin concentrations between neurons expressing WT SV2A and N573Q mutant. G) SV2A mutants harboring disrupted luminal domain 1 (SV2A-ΔL1) or L3 (SV2A-ΔL3) were expressed in SV2A/B KO neurons via lentiviral infection. Neurons were exposed to BoNT/E (100 pM) and BoNT/D (100 pM). Disrupting L1 or L3 domain did not reduce the functional entry of either BoNT/E or BoNT/D.

    Article Snippet: The following antibodies were purchased from indicated vendors: mouse monoclonal anti-HA (16B12, Covance); rabbit polyclonal anti-synapsin and guinea pig anti-vesicular glutamate transporter I (vGlut-I, Millipore) ; chicken polyclonal anti-GFP and mouse monoclonal anti-actin (Abcam).

    Techniques: Binding Assay, Transfection, Immunostaining, Construct, Infection, Expressing, Functional Assay, Mutagenesis, Incubation

    ). Blue circles represent the locations of injections of rats that received a viral vector expressing the gene of the hM4Di DREADD receptor (‘hM4Di’). Green circles represent the locations of injections of rats that received a viral vector without hM4Di receptor gene (GFP). (c) Representative photomicrograph of hM4Di-expressing dCA3 terminals in LS seen with an antibody against the HA-tag fused to the hM4Di receptor (purple/black terminals) and counterstained with neutral red. Scale bar, 100 μm. (d) Representative photomicrograph of bilateral DREADD injection into dCA3 of hippocampus (purple/black stain). As above, an antibody against the HA-tag fused to the hM4Di receptor stained the DREADDs and tissue was counterstained with a neutral red Nissl stain. Scale bar, 500 μm. (e) Inhibiting hM4Di-expressing dorsal hippocampal terminals in LS with local CNO (solid blue bars) showed a strong trend to attenuate active lever responding during context-induced, but not cue-induced, reinstatement of cocaine seeking compared to the same rats that received aCSF (open blue bars) microinjections ( n =10, effect of drug: P =0.06). Circles show individual data that overlay mean±SEM bar graphs. (f) There were no effects of CNO (solid green bars) vs aCSF (open green bars) microinjections on GFP rats during either reinstatement modality ( n =7, P > 0.05). Circles show individual data that overlay mean±SEM bar graphs.

    Journal: Neuropsychopharmacology

    Article Title: Dorsal Hippocampus Drives Context-Induced Cocaine Seeking via Inputs to Lateral Septum

    doi: 10.1038/npp.2017.144

    Figure Lengend Snippet: ). Blue circles represent the locations of injections of rats that received a viral vector expressing the gene of the hM4Di DREADD receptor (‘hM4Di’). Green circles represent the locations of injections of rats that received a viral vector without hM4Di receptor gene (GFP). (c) Representative photomicrograph of hM4Di-expressing dCA3 terminals in LS seen with an antibody against the HA-tag fused to the hM4Di receptor (purple/black terminals) and counterstained with neutral red. Scale bar, 100 μm. (d) Representative photomicrograph of bilateral DREADD injection into dCA3 of hippocampus (purple/black stain). As above, an antibody against the HA-tag fused to the hM4Di receptor stained the DREADDs and tissue was counterstained with a neutral red Nissl stain. Scale bar, 500 μm. (e) Inhibiting hM4Di-expressing dorsal hippocampal terminals in LS with local CNO (solid blue bars) showed a strong trend to attenuate active lever responding during context-induced, but not cue-induced, reinstatement of cocaine seeking compared to the same rats that received aCSF (open blue bars) microinjections ( n =10, effect of drug: P =0.06). Circles show individual data that overlay mean±SEM bar graphs. (f) There were no effects of CNO (solid green bars) vs aCSF (open green bars) microinjections on GFP rats during either reinstatement modality ( n =7, P > 0.05). Circles show individual data that overlay mean±SEM bar graphs.

    Article Snippet: Control virus brain tissue (synapsin promoter-driven GFP virus without the DREADD gene) was incubated with chicken anti-GFP primary antibody (1 : 2000; Abcam; ab13970) and donkey anti-chicken secondary antibody (1 : 500; Jackson ImmunoResearch Laboratories).

    Techniques: Plasmid Preparation, Expressing, Injection, Staining

    CNO activation of hM4Di receptors in terminals of ventral hippocampal inputs to LS does not block reinstatement. (a) The average number of Fos+ cells per brain slice in vCA1 showed a trend for more Fos+ cells during cue-induced (light blue bars; n =6) and context-induced (dark blue bars; n =8) reinstatement relative to extinction (white bars; n =5) or yoked saline groups (gray bars; n =5; one-way ANOVA, P =0.097). (b) The CA3 region of ventral hippocampus (vCA3) did not show significant differences between groups in the number of Fos+ cells ( P > 0.05). (c) Representative photomicrograph of coronal section through ventral hippocampus showing retrograde labeling after CTb injection into ipsilateral LS. Medial is to the right, dorsal is up. The black square represents the area of the magnified region of the inset. Scale bar, 500 μm. Inset shows magnified photomicrograph of neurons with or without Fos in vCA1 labeled by retrograde tracer from LS (CTb+ brown cytoplasmic stain), Fos+ (black nuclear stain), and CTb+ neurons co-labeled with Fos (Fos+CTb+, combined brown cytoplasmic stain with black nuclear stain). Scale bar, 50 μm. (d) A larger percentage of ipsilateral vCA1 neurons retrogradely labeled from LS expressed significant Fos during context-induced ( n =10, dark blue bars) and cue-induced reinstatement ( n =10, light blue bars) compared to extinction ( n =9, white bars) ( P =0.002). (e) There were no significant differences across groups in the percentages of ipsilateral vCA3 neurons retrogradely labeled from LS that expressed Fos ( P ). Blue circles represent injection sites of rats that received a vector expressing the hM4Di DREADD receptor gene (hM4Di). Green circles represent the injection sites of rats that received the same vector but contained GFP instead of the hM4Di receptor gene. (h) Representative photomicrograph of DREADD injection into ventral hippocampus as seen by a DsRed antibody against the mcherry-tag fused to the hM4Di DREADD receptor (purple/black regions) and counterstained with neutral red. Black square represents the zoomed area seen in the inset. Scale bar, 1000 μm. Inset shows cell body and axonal labeling of ventral hippocampal neurons expressing the hM4Di receptor. Inset scale bar, 100 μm. Whole images adjusted for brightness. (i) Microinjection of CNO among hM4Di-expressing ventral hippocampal terminals in LS ( n =8, solid blue bars) did not affect active lever responding during context-induced or cue-induced reinstatement of cocaine seeking compared to the same rats that received aCSF microinjections (open blue bars, P > 0.05; two-way RM ANOVA). Individual data overlay mean±SEM bar graphs. (j) There were also no effects of CNO (solid green bars) vs aCSF (open green bars) microinjections on rats without the DREADD receptor (GFP controls) during context-induced or cue-induced reinstatement of cocaine seeking ( n =6; P > 0.05). Circles show individual data that overlay mean±SEM bar graphs.

    Journal: Neuropsychopharmacology

    Article Title: Dorsal Hippocampus Drives Context-Induced Cocaine Seeking via Inputs to Lateral Septum

    doi: 10.1038/npp.2017.144

    Figure Lengend Snippet: CNO activation of hM4Di receptors in terminals of ventral hippocampal inputs to LS does not block reinstatement. (a) The average number of Fos+ cells per brain slice in vCA1 showed a trend for more Fos+ cells during cue-induced (light blue bars; n =6) and context-induced (dark blue bars; n =8) reinstatement relative to extinction (white bars; n =5) or yoked saline groups (gray bars; n =5; one-way ANOVA, P =0.097). (b) The CA3 region of ventral hippocampus (vCA3) did not show significant differences between groups in the number of Fos+ cells ( P > 0.05). (c) Representative photomicrograph of coronal section through ventral hippocampus showing retrograde labeling after CTb injection into ipsilateral LS. Medial is to the right, dorsal is up. The black square represents the area of the magnified region of the inset. Scale bar, 500 μm. Inset shows magnified photomicrograph of neurons with or without Fos in vCA1 labeled by retrograde tracer from LS (CTb+ brown cytoplasmic stain), Fos+ (black nuclear stain), and CTb+ neurons co-labeled with Fos (Fos+CTb+, combined brown cytoplasmic stain with black nuclear stain). Scale bar, 50 μm. (d) A larger percentage of ipsilateral vCA1 neurons retrogradely labeled from LS expressed significant Fos during context-induced ( n =10, dark blue bars) and cue-induced reinstatement ( n =10, light blue bars) compared to extinction ( n =9, white bars) ( P =0.002). (e) There were no significant differences across groups in the percentages of ipsilateral vCA3 neurons retrogradely labeled from LS that expressed Fos ( P ). Blue circles represent injection sites of rats that received a vector expressing the hM4Di DREADD receptor gene (hM4Di). Green circles represent the injection sites of rats that received the same vector but contained GFP instead of the hM4Di receptor gene. (h) Representative photomicrograph of DREADD injection into ventral hippocampus as seen by a DsRed antibody against the mcherry-tag fused to the hM4Di DREADD receptor (purple/black regions) and counterstained with neutral red. Black square represents the zoomed area seen in the inset. Scale bar, 1000 μm. Inset shows cell body and axonal labeling of ventral hippocampal neurons expressing the hM4Di receptor. Inset scale bar, 100 μm. Whole images adjusted for brightness. (i) Microinjection of CNO among hM4Di-expressing ventral hippocampal terminals in LS ( n =8, solid blue bars) did not affect active lever responding during context-induced or cue-induced reinstatement of cocaine seeking compared to the same rats that received aCSF microinjections (open blue bars, P > 0.05; two-way RM ANOVA). Individual data overlay mean±SEM bar graphs. (j) There were also no effects of CNO (solid green bars) vs aCSF (open green bars) microinjections on rats without the DREADD receptor (GFP controls) during context-induced or cue-induced reinstatement of cocaine seeking ( n =6; P > 0.05). Circles show individual data that overlay mean±SEM bar graphs.

    Article Snippet: Control virus brain tissue (synapsin promoter-driven GFP virus without the DREADD gene) was incubated with chicken anti-GFP primary antibody (1 : 2000; Abcam; ab13970) and donkey anti-chicken secondary antibody (1 : 500; Jackson ImmunoResearch Laboratories).

    Techniques: Activation Assay, Blocking Assay, Slice Preparation, Labeling, CtB Assay, Injection, Staining, Plasmid Preparation, Expressing

    The embryonic POA gives rise to cells that migrate to the cortex. A , Schema of the experimental design followed in B–E′ . B , B′ , A representative case of the distribution of GFP ( B )- and Nkx2-1 ( B′ )-expressing cells in a coronal section through the telencephalon of an E15.5 embryo in which the POA was electroporated at E12.5. The white arrowhead indicates the location of basal forebrain cells derived from the POA. C , C′ , High-magnification images of the boxed areas shown in B and B′ , respectively. The open arrowheads in B and C mark labeled cells with migratory morphology in the subpallium and in the cortex. D , D′ , E , E′ , Images of representative cells found in the cortex. These cells typically stain for Calbindin ( D , D′ ) and do not express Lhx6 ( E , E′ ). Scale bars: (in B ) B , B′ , 250 μm; (in C ) C , C′ , 100 μm; (in D ) D , D′ , E , E′ , 25 μm.

    Journal: The Journal of Neuroscience

    Article Title: The Embryonic Preoptic Area Is a Novel Source of Cortical GABAergic Interneurons

    doi: 10.1523/JNEUROSCI.0604-09.2009

    Figure Lengend Snippet: The embryonic POA gives rise to cells that migrate to the cortex. A , Schema of the experimental design followed in B–E′ . B , B′ , A representative case of the distribution of GFP ( B )- and Nkx2-1 ( B′ )-expressing cells in a coronal section through the telencephalon of an E15.5 embryo in which the POA was electroporated at E12.5. The white arrowhead indicates the location of basal forebrain cells derived from the POA. C , C′ , High-magnification images of the boxed areas shown in B and B′ , respectively. The open arrowheads in B and C mark labeled cells with migratory morphology in the subpallium and in the cortex. D , D′ , E , E′ , Images of representative cells found in the cortex. These cells typically stain for Calbindin ( D , D′ ) and do not express Lhx6 ( E , E′ ). Scale bars: (in B ) B , B′ , 250 μm; (in C ) C , C′ , 100 μm; (in D ) D , D′ , E , E′ , 25 μm.

    Article Snippet: The following primary antibodies were used: rat anti-5-bromo-2′-deoxyuridine (BrdU) (1:100; Accurate), mouse anti-Cre (1:2000; a gift from G. Schütz), rabbit anti-CyclinD1 (1:1; Thermo Fisher Scientific), rabbit anti-GABA (1:1000; Sigma-Aldrich), chicken anti-green fluorescent protein (GFP) (1:3000; Aves Labs), rabbit anti-Lhx6 (1:1000; a gift from V. Pachnis), rabbit anti-NG2 (1:200; Millipore Bioscience Research Reagents), rabbit anti-Nkx2-1 (1:2000; Biopat), mouse anti-neuronal nuclei (NeuN) (1:500; Millipore Bioscience Research Reagents), rabbit anti-neuronal isoform of nitric oxide synthase (nNOS) (1:1000; Immunostar), rabbit anti-PH3 (1:500; Millipore), rabbit anti-parvalbumin (PV) (1:5000; Swant), rat anti-somatostatin (SST) (1:200; Millipore Bioscience Research Reagents), rabbit anti-S100 (1:1000; Sigma-Aldrich), rabbit anti-calretinin (CR) (1:3000; Swant), and rabbit anti-vasointestinal peptide (VIP) (1:1000; Immunostar).

    Techniques: Expressing, Derivative Assay, Labeling, Staining