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  • 85
    Thermo Fisher gene exp slc18a3 rn00581454 s1
    Gene Exp Slc18a3 Rn00581454 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Novus Biologicals antibody against vacht
    Antibody Against Vacht, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Antibodies Inc vacht
    Antibodies against <t>VAChT,</t> VNUT, VGLUT1 and <t>VGLUT2</t> stain axons and presynaptic boutons residing within the electric organ. (A) Cartoon depicting the electric organ of Torpedo californica (1 side of the paired organ shown) and its innervation by four electromotor nerves that project from the electric lobe of the central nervous system (green). From the surface, the electroplaque cells of the electric lobe appear as a honeycomb. Viewed from the side the electroplaque cells appear as large, pancake‐like stacks. Each individual electroplaque has a top surface covered in nicotinic acetylcholine receptors (red surface). That surface is richly innervated with presynaptic boutons and axons. Axons run in between the pancake stacks before entering the target electroplaque and forming synapses. The drawing to the far right in A illustrates the view shown in the remainder of the figure; however, in the immunostains, the receptor‐rich surface of each electroplaque commonly runs at an angle to the central core containing only axons. (B–H) Labeling with each of the neurotransmitter transporter antibodies was done in conjunction with α ‐Bungarotoxin to mark the postsynaptic surface on the electroplaque cells. (B) VAChT polyclonal antibody, and (C) VNUT polyclonal antibody. (D) Application of a secondary antibody to tissue to which no primary polyclonal was applied. (E) VAChT monoclonal antibody, (F) VGLUT1 monoclonal antibody (G) VGLUT2 monoclonal antibody, and (H) secondary antibody in a control to which no primary monoclonal antibody was applied. Axons and presynaptic labeling can be seen in (B, C, and E–G) but no appreciable label was detected in the controls (D and H). Scale bar = 5 microns.
    Vacht, supplied by Antibodies Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    vacht - by Bioz Stars, 2020-08
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    85
    Millipore slc18a3
    mESC-derived MNs form cholinergic synapses with muscle cells in vitro. Immunofluorescence analysis of proteins expressed at nerve terminals of mESC-derived MNs. The three columns show: (A, D, G, J, M, P) brightfield images of axon terminals contacting muscle cells; (B, E, H, K, N, Q) green fluorescence associated with the Hb9::EGFP MN reporter; (C, F, I, L, O, R) red fluorescence corresponding to antibody staining for the indicated presynaptic proteins: <t>Slc18a3</t> (VAChT), Slc5a7 (ChT1), Syntaxin 1a (Stx1a), Snap25, Sv2, and Synaptophysin (Syp). Scale bar is 20 µm.
    Slc18a3, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega slc18a3
    Differentiation Defects in Rb KO SACs (A) P18 horizontal sections of WT, Rb KO, and Rb/E2f1 DKO retina were stained for nuclei (DAPI, blue), Calb2 (green), and <t>Slc18a3</t> (red). (B) Confocal images of P30 horizontal sections of WT and Rb KO retina were stained for nuclei (DAPI, blue), Chat and Slc18a3 (both red), and Camk2a (green). In the Rb KO section, the red stain is Chat only, as Slc18a3 is missing (see [A]). (C) Quantification of dense Calb2 + cell bodies in the INL, total Slc18a3 + cell bodies, and Camk2a + cell bodies in the INL. Error bars represent SD of measurements from three animals, and asterisks indicate significant differences between retinas of WT and the indicated genotypes (*, p
    Slc18a3, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore α vacht
    Bilateral superior cervical ganglionectomy in SHR attenuates sympathetic fibre innervation of vertebrobasilar arteries but is without effect on their remodelling A , representative images of vertebrobasilar arteries showing immunofluorescence staining α‐DBH‐AF594 and <t>α‐VAChT‐AF488</t> after SCGx or sham operation in SHR and corresponding bright field (BF) images. The arrowheads indicate exemplar sympathetic fibres stained with DBH antibody. B , the reduction in DBH staining is clearly evident after ganglionectomy. Percentage change in sympathetic (DBH positive) and parasympathetic (VAChT positive) fibre densities in SCGx compared to sham animals according to area. Significant differences to normalised sham values are indicated on the graph: * P
    α Vacht, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology vacht peptide
    Bilateral superior cervical ganglionectomy in SHR attenuates sympathetic fibre innervation of vertebrobasilar arteries but is without effect on their remodelling A , representative images of vertebrobasilar arteries showing immunofluorescence staining α‐DBH‐AF594 and <t>α‐VAChT‐AF488</t> after SCGx or sham operation in SHR and corresponding bright field (BF) images. The arrowheads indicate exemplar sympathetic fibres stained with DBH antibody. B , the reduction in DBH staining is clearly evident after ganglionectomy. Percentage change in sympathetic (DBH positive) and parasympathetic (VAChT positive) fibre densities in SCGx compared to sham animals according to area. Significant differences to normalised sham values are indicated on the graph: * P
    Vacht Peptide, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    SLIT2 LTD slc18a3
    Loss of Shox2 function in the facial motor nucleus interferes with the proper development of the medial subnuclei. a Diagram of a coronal section through the P0 brain highlights the region represented in panels b – v , particularly the 7 neuronal clusters that form the facial motor nucleus ( nVII , dashed-circle ) (image adapted from [ 23 ]). b X-gal stained coronal sections through P0 brains shows a loss of medial neuronal clusters in the facial motor nucleus of Nestin - Cre; Shox2 lacZ/flox ( B ″) brains as compared to controls ( B ′). c – v ISH on P0 control ( c , e , g , i , k , m , o , q , s , u ) and Nestin - Cre; Shox2 flox/ − ( d , f , h , j , l , n , p , r , t , v ) coronal sections shows loss of Shox2 expression (compare c to d , dashed-circle ), and decreases in Isl1 (compare e to f , dashed-circle ), Phox2b (compare g to h , dashed-circle ), Cntn2 (compare i to j , dashed-circle ), Tubb3 (compare k to l , dashed-circle ), Periph (compare m to n , dashed-circle ), <t>Slc18a3</t> (compare o to p , dashed-circle ), Slit2 (compare q to r , dashed-circle ), Shh (compare s to t , dashed-circle ) and Ptch1 (compare u to v , dashed-circle ) expression in the facial motor nucleus. DA dorsal accessory, DM dorsomedial, VM ventromedial, DI dorsal intermediate, VI ventral intermediate, DL dorsolateral, Lat lateral. Scale bar 250 μm.
    Slc18a3, supplied by SLIT2 LTD, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher gene exp slc18a3 hs00268179 s1
    Loss of Shox2 function in the facial motor nucleus interferes with the proper development of the medial subnuclei. a Diagram of a coronal section through the P0 brain highlights the region represented in panels b – v , particularly the 7 neuronal clusters that form the facial motor nucleus ( nVII , dashed-circle ) (image adapted from [ 23 ]). b X-gal stained coronal sections through P0 brains shows a loss of medial neuronal clusters in the facial motor nucleus of Nestin - Cre; Shox2 lacZ/flox ( B ″) brains as compared to controls ( B ′). c – v ISH on P0 control ( c , e , g , i , k , m , o , q , s , u ) and Nestin - Cre; Shox2 flox/ − ( d , f , h , j , l , n , p , r , t , v ) coronal sections shows loss of Shox2 expression (compare c to d , dashed-circle ), and decreases in Isl1 (compare e to f , dashed-circle ), Phox2b (compare g to h , dashed-circle ), Cntn2 (compare i to j , dashed-circle ), Tubb3 (compare k to l , dashed-circle ), Periph (compare m to n , dashed-circle ), <t>Slc18a3</t> (compare o to p , dashed-circle ), Slit2 (compare q to r , dashed-circle ), Shh (compare s to t , dashed-circle ) and Ptch1 (compare u to v , dashed-circle ) expression in the facial motor nucleus. DA dorsal accessory, DM dorsomedial, VM ventromedial, DI dorsal intermediate, VI ventral intermediate, DL dorsolateral, Lat lateral. Scale bar 250 μm.
    Gene Exp Slc18a3 Hs00268179 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher gene exp slc18a3 mm00491465 s1
    ChIP-seq assays revealed Isl-Lhx3-hexamer-binding sites in a cholinergic gene battery. ( A ) Schematic representation of cholinergic neurotransmission system. Acly, ATP-citrate lyase; CoA, coenzyme A; ChAT, choline acetyltransferase; ACh, acetylcholine; <t>VAChT,</t> vesicular acetylcholine transporter; AChE, Acetylcholine esterase; CHT, high affinity choline transporter; AChRs, Acetylcholine receptors. ( B ) ChIP-seq tag profile of the genomic region surrounding a battery of cholinergic genes <t>ChAT/VAChT</t> , CHT , and Acly loci. Each cholinergic gene is indicated, and the blue arrows represent the direction of transcription. Mam cons., mammalian conservation. The ChIP-seq data was deposited in the GEO database (assession no. GSE50993) [20] . ( C ) Schematic representation of the location of the HxRE motifs in each of the 500 bp-long cholinergic gene peaks. The number shows the relative position within the peak (0, the center position of each peak). ( D ) In vivo ChIP assays in dissected E12.5 embryonic spinal cords to monitor the binding of the Isl1-Lhx3-hexamer to the cholinergic enhancers. Schematic representation of the ChAT gene is shown on the top. The arrows indicate two sets of primers detecting ChAT -enhancer ( ChAT -enh) and a negative control region lacking the Isl1-Lhx3-binding peak ( ChAT -neg). Isl1, Lhx3, and NLI were recruited to the cholinergic enhancers in embryonic spinal cords. Error bars indicate standard deviation.
    Gene Exp Slc18a3 Mm00491465 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology anti vacht
    Human detrusor ICCs. A , human detrusor labeled with anti-SM myosin ( SMM ). B , at mucosa-detrusor interface SMCs were arranged in loose network, which was organized into distinct bundles in detrusor muscularis. C to E , representative human detrusor co-labeled with anti-SM myosin and anti- <t>KIT</t> (green areas). Elongated, branched KIT positive ICC track SM bundles and occupy spaces between bundles with obvious spiky morphology. E , nuclei counterstained with 4,6-diamidino-2-phenylindole (blue areas). F , detrusor ICCs (green areas) associated with <t>vAChT</t> nerves (red areas).
    Anti Vacht, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Enzo Biochem vacht
    Human detrusor ICCs. A , human detrusor labeled with anti-SM myosin ( SMM ). B , at mucosa-detrusor interface SMCs were arranged in loose network, which was organized into distinct bundles in detrusor muscularis. C to E , representative human detrusor co-labeled with anti-SM myosin and anti- <t>KIT</t> (green areas). Elongated, branched KIT positive ICC track SM bundles and occupy spaces between bundles with obvious spiky morphology. E , nuclei counterstained with 4,6-diamidino-2-phenylindole (blue areas). F , detrusor ICCs (green areas) associated with <t>vAChT</t> nerves (red areas).
    Vacht, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Synaptic Systems vacht
    Effects of <t>CHAT</t> knockdown in medial habenula on depression-like behaviors. ( a ) Schematic representation of the AAV vector engineered to induce CHAT knockdown. ( b ) Experimental paradigm for behavioral testing of rats infected with the virus. ( c ) Representative photomicrographs of Hb slices from the rats injected with AAV-sh-SCR (control) or AAV-sh-CHAT (CHAT knockdown). CHAT protein expression was visualized as red immunofluorescence. Cell nuclei and viral infection were visualized using Hoechst staining (blue) and GFP (green), respectively. The inset box shows a higher magnification of an infected MHb region. None of the neurons infected with the AAV-sh-CHAT showed CHAT expression. Scale bar, 50 μm. ( d ) <t>VACHT</t> expression is not affected by AAV-sh-CHAT. Hb neurons injected with AAV-sh-CHAT were immunostained for VACHT (red) and EGFP (green). Scale bar, 50 μm. ( e ) CHAT and VACHT western blots from the Hb of rats injected with AAV-sh-CHAT. Rats were injected with AAV-sh-SCR (control) or AAV-sh-CHAT, which were expressed for 3 weeks before western blot analysis. ( f ) Quantification of the effects of shRNA-mediated CHAT knockdown. Data represent mean ± SEM (sh-SCR, n = 4 rats; sh-CHAT, n = 4; * P
    Vacht, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 89/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega anti vacht
    Molecular distinctions among αRGCs. A, B : Retinal whole mounts were stained with antibodies to Opn (green), plus antibodies to one of 3 POU-domain transcription factors (Brn3a, Brn3b or Brn3c; red in A ) or one of 3 calcium binding proteins (parvalbumin [PV], <t>calbindin</t> or calretinin; red in B ). Brn3b and PV mark most αRGCs whereas Brn3a, Brn3c and calbindin mark subsets; most αRGCs are calretinin-negative. C: Whole mounts of YFP-H retina were quadruply stained for YFP, Opn, <t>vAChT</t> and the indicated marker. Cells that were Opn, YFP, and marker triple-positive were identified (green, cyan and red, respectively in top panels) and imaged (YFP only, middle panels). Stratification of YFP-positive dendrites was then determined with reference to that of starburst amacrines (vAChT-positive, red in bottom panels). Arrows point to the same cell displayed in each panel. Results are representative of 7–10 cells per type from 5 mice. Scale bar = 50 μm.
    Anti Vacht, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher vacht sequence
    Molecular distinctions among αRGCs. A, B : Retinal whole mounts were stained with antibodies to Opn (green), plus antibodies to one of 3 POU-domain transcription factors (Brn3a, Brn3b or Brn3c; red in A ) or one of 3 calcium binding proteins (parvalbumin [PV], <t>calbindin</t> or calretinin; red in B ). Brn3b and PV mark most αRGCs whereas Brn3a, Brn3c and calbindin mark subsets; most αRGCs are calretinin-negative. C: Whole mounts of YFP-H retina were quadruply stained for YFP, Opn, <t>vAChT</t> and the indicated marker. Cells that were Opn, YFP, and marker triple-positive were identified (green, cyan and red, respectively in top panels) and imaged (YFP only, middle panels). Stratification of YFP-positive dendrites was then determined with reference to that of starburst amacrines (vAChT-positive, red in bottom panels). Arrows point to the same cell displayed in each panel. Results are representative of 7–10 cells per type from 5 mice. Scale bar = 50 μm.
    Vacht Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    vacht  (Abcam)
    89
    Abcam vacht
    Reconstitution of the mast cell pool in the lungs of Kit W-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. ( a ) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. ( b ) Representative images of toluidine blue staining of mast cells in the lungs of Kit W-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in Kit W-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm 2 ) in mid-lobe sections of each mouse lung and 4 mice for each condition. ( c ) Representative images of TuJ1 staining of airways (0.1–0.3 mm 2 in luminal area) from PBS- and OVA-exposed Kit W-sh/W-sh mice that received intra-tracheal instillation of WT or NT4 −/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed Kit W-sh/W-sh mice with and without adoptive transfer of WT and NT4 −/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. ( d ) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA-exposed wild type mice were assayed for the levels of <t>VAChT.</t> Each lane represents 1 mouse. <t>GAPDH</t> was loading control. Data were normalized to PBS control mice. N=12. ( e ) Western blot analysis for cholinergic innervation in lungs of Kit W-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed Kit W-sh/W-sh mice. ( f ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and Kit W-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and Kit W-sh/W-sh mice following OVA exposure were marked. ( g ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed Kit W-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Statistically significant differences between WT and NT4 −/− mast cell transfer were marked. The same results of OVA-exposed Kit W-sh/W-sh mice were plotted in both ( f ) and ( g ). *P
    Vacht, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore rat vacht
    Reconstitution of the mast cell pool in the lungs of Kit W-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. ( a ) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. ( b ) Representative images of toluidine blue staining of mast cells in the lungs of Kit W-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in Kit W-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm 2 ) in mid-lobe sections of each mouse lung and 4 mice for each condition. ( c ) Representative images of TuJ1 staining of airways (0.1–0.3 mm 2 in luminal area) from PBS- and OVA-exposed Kit W-sh/W-sh mice that received intra-tracheal instillation of WT or NT4 −/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed Kit W-sh/W-sh mice with and without adoptive transfer of WT and NT4 −/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. ( d ) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA-exposed wild type mice were assayed for the levels of <t>VAChT.</t> Each lane represents 1 mouse. <t>GAPDH</t> was loading control. Data were normalized to PBS control mice. N=12. ( e ) Western blot analysis for cholinergic innervation in lungs of Kit W-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed Kit W-sh/W-sh mice. ( f ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and Kit W-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and Kit W-sh/W-sh mice following OVA exposure were marked. ( g ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed Kit W-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Statistically significant differences between WT and NT4 −/− mast cell transfer were marked. The same results of OVA-exposed Kit W-sh/W-sh mice were plotted in both ( f ) and ( g ). *P
    Rat Vacht, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore polyclonal vacht antiserum
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    Reconstitution of the mast cell pool in the lungs of Kit W-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. ( a ) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. ( b ) Representative images of toluidine blue staining of mast cells in the lungs of Kit W-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in Kit W-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm 2 ) in mid-lobe sections of each mouse lung and 4 mice for each condition. ( c ) Representative images of TuJ1 staining of airways (0.1–0.3 mm 2 in luminal area) from PBS- and OVA-exposed Kit W-sh/W-sh mice that received intra-tracheal instillation of WT or NT4 −/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed Kit W-sh/W-sh mice with and without adoptive transfer of WT and NT4 −/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. ( d ) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA-exposed wild type mice were assayed for the levels of <t>VAChT.</t> Each lane represents 1 mouse. <t>GAPDH</t> was loading control. Data were normalized to PBS control mice. N=12. ( e ) Western blot analysis for cholinergic innervation in lungs of Kit W-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed Kit W-sh/W-sh mice. ( f ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and Kit W-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and Kit W-sh/W-sh mice following OVA exposure were marked. ( g ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed Kit W-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Statistically significant differences between WT and NT4 −/− mast cell transfer were marked. The same results of OVA-exposed Kit W-sh/W-sh mice were plotted in both ( f ) and ( g ). *P
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    Reconstitution of the mast cell pool in the lungs of Kit W-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. ( a ) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. ( b ) Representative images of toluidine blue staining of mast cells in the lungs of Kit W-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in Kit W-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm 2 ) in mid-lobe sections of each mouse lung and 4 mice for each condition. ( c ) Representative images of TuJ1 staining of airways (0.1–0.3 mm 2 in luminal area) from PBS- and OVA-exposed Kit W-sh/W-sh mice that received intra-tracheal instillation of WT or NT4 −/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed Kit W-sh/W-sh mice with and without adoptive transfer of WT and NT4 −/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. ( d ) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA-exposed wild type mice were assayed for the levels of <t>VAChT.</t> Each lane represents 1 mouse. <t>GAPDH</t> was loading control. Data were normalized to PBS control mice. N=12. ( e ) Western blot analysis for cholinergic innervation in lungs of Kit W-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed Kit W-sh/W-sh mice. ( f ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and Kit W-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and Kit W-sh/W-sh mice following OVA exposure were marked. ( g ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed Kit W-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Statistically significant differences between WT and NT4 −/− mast cell transfer were marked. The same results of OVA-exposed Kit W-sh/W-sh mice were plotted in both ( f ) and ( g ). *P
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    Reconstitution of the mast cell pool in the lungs of Kit W-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. ( a ) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. ( b ) Representative images of toluidine blue staining of mast cells in the lungs of Kit W-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in Kit W-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm 2 ) in mid-lobe sections of each mouse lung and 4 mice for each condition. ( c ) Representative images of TuJ1 staining of airways (0.1–0.3 mm 2 in luminal area) from PBS- and OVA-exposed Kit W-sh/W-sh mice that received intra-tracheal instillation of WT or NT4 −/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed Kit W-sh/W-sh mice with and without adoptive transfer of WT and NT4 −/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. ( d ) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA-exposed wild type mice were assayed for the levels of <t>VAChT.</t> Each lane represents 1 mouse. <t>GAPDH</t> was loading control. Data were normalized to PBS control mice. N=12. ( e ) Western blot analysis for cholinergic innervation in lungs of Kit W-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed Kit W-sh/W-sh mice. ( f ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and Kit W-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and Kit W-sh/W-sh mice following OVA exposure were marked. ( g ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed Kit W-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Statistically significant differences between WT and NT4 −/− mast cell transfer were marked. The same results of OVA-exposed Kit W-sh/W-sh mice were plotted in both ( f ) and ( g ). *P
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    Image Search Results


    Antibodies against VAChT, VNUT, VGLUT1 and VGLUT2 stain axons and presynaptic boutons residing within the electric organ. (A) Cartoon depicting the electric organ of Torpedo californica (1 side of the paired organ shown) and its innervation by four electromotor nerves that project from the electric lobe of the central nervous system (green). From the surface, the electroplaque cells of the electric lobe appear as a honeycomb. Viewed from the side the electroplaque cells appear as large, pancake‐like stacks. Each individual electroplaque has a top surface covered in nicotinic acetylcholine receptors (red surface). That surface is richly innervated with presynaptic boutons and axons. Axons run in between the pancake stacks before entering the target electroplaque and forming synapses. The drawing to the far right in A illustrates the view shown in the remainder of the figure; however, in the immunostains, the receptor‐rich surface of each electroplaque commonly runs at an angle to the central core containing only axons. (B–H) Labeling with each of the neurotransmitter transporter antibodies was done in conjunction with α ‐Bungarotoxin to mark the postsynaptic surface on the electroplaque cells. (B) VAChT polyclonal antibody, and (C) VNUT polyclonal antibody. (D) Application of a secondary antibody to tissue to which no primary polyclonal was applied. (E) VAChT monoclonal antibody, (F) VGLUT1 monoclonal antibody (G) VGLUT2 monoclonal antibody, and (H) secondary antibody in a control to which no primary monoclonal antibody was applied. Axons and presynaptic labeling can be seen in (B, C, and E–G) but no appreciable label was detected in the controls (D and H). Scale bar = 5 microns.

    Journal: Physiological Reports

    Article Title: Individual synaptic vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP

    doi: 10.1002/phy2.206

    Figure Lengend Snippet: Antibodies against VAChT, VNUT, VGLUT1 and VGLUT2 stain axons and presynaptic boutons residing within the electric organ. (A) Cartoon depicting the electric organ of Torpedo californica (1 side of the paired organ shown) and its innervation by four electromotor nerves that project from the electric lobe of the central nervous system (green). From the surface, the electroplaque cells of the electric lobe appear as a honeycomb. Viewed from the side the electroplaque cells appear as large, pancake‐like stacks. Each individual electroplaque has a top surface covered in nicotinic acetylcholine receptors (red surface). That surface is richly innervated with presynaptic boutons and axons. Axons run in between the pancake stacks before entering the target electroplaque and forming synapses. The drawing to the far right in A illustrates the view shown in the remainder of the figure; however, in the immunostains, the receptor‐rich surface of each electroplaque commonly runs at an angle to the central core containing only axons. (B–H) Labeling with each of the neurotransmitter transporter antibodies was done in conjunction with α ‐Bungarotoxin to mark the postsynaptic surface on the electroplaque cells. (B) VAChT polyclonal antibody, and (C) VNUT polyclonal antibody. (D) Application of a secondary antibody to tissue to which no primary polyclonal was applied. (E) VAChT monoclonal antibody, (F) VGLUT1 monoclonal antibody (G) VGLUT2 monoclonal antibody, and (H) secondary antibody in a control to which no primary monoclonal antibody was applied. Axons and presynaptic labeling can be seen in (B, C, and E–G) but no appreciable label was detected in the controls (D and H). Scale bar = 5 microns.

    Article Snippet: Reagents Mouse monoclonal antibodies to VGLUT1 (N28/9), VGLUT2 (N29/29), VGLUT3 (N34/34), as well as the mouse monoclonal antibody for VAChT (N6/38) were purchased from Antibodies Incorporated (UC Davis/NIH NeuroMab Facility, Davis, CA).

    Techniques: Staining, Labeling

    Synaptic vesicles can be enriched from electroplaques of Torpedo californica and shown to contain four neurotransmitter transporters. (A) Electron micrograph image (30,000×) of negatively stained vesicles isolated and enriched from electroplaque tissue. Sample contains abundant ~80 nm vesicles (some marked with single arrowheads) and occasional large clusters of vesicles and debris (double arrowheads). (A) ( insert ) Higher magnification (50,000×) negative stain image shows characteristic profile of membrane vesicles, Scale bar = 100 nm. (B) Immunoblot of the 38 kDa protein synaptophysin demonstrates isolation and enrichment of synaptic vesicles during the isolation procedure. Synaptophysin was seen in the tissue (T), and discarded pellets (P1, and P2), however, during isolation the majority of vesicles are maintained in the much larger by volume supernatant (S1 and S2) until the final centrifugation. In the final centrifugation, the vesicles move through the supernatant (S3) and collect on the fluffy layer (FL). The fluffy layer was collected, and the synaptic vesicles were further enriched by size exclusion chromatography (SV). Purified vesicles were tested by immunoblot and found to be positive for the ~60 kDa transporter proteins: VAChT, VNUT, VGLUT1, VGLUT2. Extra bands are possibly due to differences in protein glycosylation. No signal was detected for the transporter protein VGLUT3.

    Journal: Physiological Reports

    Article Title: Individual synaptic vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP

    doi: 10.1002/phy2.206

    Figure Lengend Snippet: Synaptic vesicles can be enriched from electroplaques of Torpedo californica and shown to contain four neurotransmitter transporters. (A) Electron micrograph image (30,000×) of negatively stained vesicles isolated and enriched from electroplaque tissue. Sample contains abundant ~80 nm vesicles (some marked with single arrowheads) and occasional large clusters of vesicles and debris (double arrowheads). (A) ( insert ) Higher magnification (50,000×) negative stain image shows characteristic profile of membrane vesicles, Scale bar = 100 nm. (B) Immunoblot of the 38 kDa protein synaptophysin demonstrates isolation and enrichment of synaptic vesicles during the isolation procedure. Synaptophysin was seen in the tissue (T), and discarded pellets (P1, and P2), however, during isolation the majority of vesicles are maintained in the much larger by volume supernatant (S1 and S2) until the final centrifugation. In the final centrifugation, the vesicles move through the supernatant (S3) and collect on the fluffy layer (FL). The fluffy layer was collected, and the synaptic vesicles were further enriched by size exclusion chromatography (SV). Purified vesicles were tested by immunoblot and found to be positive for the ~60 kDa transporter proteins: VAChT, VNUT, VGLUT1, VGLUT2. Extra bands are possibly due to differences in protein glycosylation. No signal was detected for the transporter protein VGLUT3.

    Article Snippet: Reagents Mouse monoclonal antibodies to VGLUT1 (N28/9), VGLUT2 (N29/29), VGLUT3 (N34/34), as well as the mouse monoclonal antibody for VAChT (N6/38) were purchased from Antibodies Incorporated (UC Davis/NIH NeuroMab Facility, Davis, CA).

    Techniques: Staining, Isolation, Centrifugation, Size-exclusion Chromatography, Purification

    Single‐synaptic vesicle observations of vesicular transporters (VNUT, VGLUT1, or VGLUT2) colabeled with VAChT, and confidence intervals of statistically paired transporters calculated using two‐sided Agresti‐Coull confidence limits.

    Journal: Physiological Reports

    Article Title: Individual synaptic vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP

    doi: 10.1002/phy2.206

    Figure Lengend Snippet: Single‐synaptic vesicle observations of vesicular transporters (VNUT, VGLUT1, or VGLUT2) colabeled with VAChT, and confidence intervals of statistically paired transporters calculated using two‐sided Agresti‐Coull confidence limits.

    Article Snippet: Reagents Mouse monoclonal antibodies to VGLUT1 (N28/9), VGLUT2 (N29/29), VGLUT3 (N34/34), as well as the mouse monoclonal antibody for VAChT (N6/38) were purchased from Antibodies Incorporated (UC Davis/NIH NeuroMab Facility, Davis, CA).

    Techniques:

    TIRF microscope images of single synaptic vesicles. During TIRF illumination, the excitation beam travels off‐axis through the microscope objective causing the beam's incident angle at the coverslip to be too shallow to allow the beam to pass into the sample, thus leading to the total internal reflection of the beam back into the objective. An evanescent field of illumination, approximately 100 nm in depth, is created at the surface of the coverslip, allowing for selective illumination of single molecules residing on the coverslip. A diagram of expected observations could include singly label vesicles A, B, or dually labeled vesicles C . FM4‐64 was used in order to control for three possible scenarios: (D) Nonlabeled vesicles or debris, (E–G) fluorescent secondary on the coverslip not associated with any synaptic vesicles, or (H) spots that contained brighter than standard labeling in the FM4‐64 channel, as discussed in Figure 3 B, and were thus unlikely to be single vesicle events. Example TIRF images of dual labeled, single vesicle events for (I) VAChT and VNUT, (J) VAChT and VGLUT1, and (K) VAChT and VGLUT2. Scale bar = 1 micron.

    Journal: Physiological Reports

    Article Title: Individual synaptic vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP

    doi: 10.1002/phy2.206

    Figure Lengend Snippet: TIRF microscope images of single synaptic vesicles. During TIRF illumination, the excitation beam travels off‐axis through the microscope objective causing the beam's incident angle at the coverslip to be too shallow to allow the beam to pass into the sample, thus leading to the total internal reflection of the beam back into the objective. An evanescent field of illumination, approximately 100 nm in depth, is created at the surface of the coverslip, allowing for selective illumination of single molecules residing on the coverslip. A diagram of expected observations could include singly label vesicles A, B, or dually labeled vesicles C . FM4‐64 was used in order to control for three possible scenarios: (D) Nonlabeled vesicles or debris, (E–G) fluorescent secondary on the coverslip not associated with any synaptic vesicles, or (H) spots that contained brighter than standard labeling in the FM4‐64 channel, as discussed in Figure 3 B, and were thus unlikely to be single vesicle events. Example TIRF images of dual labeled, single vesicle events for (I) VAChT and VNUT, (J) VAChT and VGLUT1, and (K) VAChT and VGLUT2. Scale bar = 1 micron.

    Article Snippet: Reagents Mouse monoclonal antibodies to VGLUT1 (N28/9), VGLUT2 (N29/29), VGLUT3 (N34/34), as well as the mouse monoclonal antibody for VAChT (N6/38) were purchased from Antibodies Incorporated (UC Davis/NIH NeuroMab Facility, Davis, CA).

    Techniques: Microscopy, Labeling

    mESC-derived MNs form cholinergic synapses with muscle cells in vitro. Immunofluorescence analysis of proteins expressed at nerve terminals of mESC-derived MNs. The three columns show: (A, D, G, J, M, P) brightfield images of axon terminals contacting muscle cells; (B, E, H, K, N, Q) green fluorescence associated with the Hb9::EGFP MN reporter; (C, F, I, L, O, R) red fluorescence corresponding to antibody staining for the indicated presynaptic proteins: Slc18a3 (VAChT), Slc5a7 (ChT1), Syntaxin 1a (Stx1a), Snap25, Sv2, and Synaptophysin (Syp). Scale bar is 20 µm.

    Journal: PLoS ONE

    Article Title: Functional Neuromuscular Junctions Formed by Embryonic Stem Cell-Derived Motor Neurons

    doi: 10.1371/journal.pone.0036049

    Figure Lengend Snippet: mESC-derived MNs form cholinergic synapses with muscle cells in vitro. Immunofluorescence analysis of proteins expressed at nerve terminals of mESC-derived MNs. The three columns show: (A, D, G, J, M, P) brightfield images of axon terminals contacting muscle cells; (B, E, H, K, N, Q) green fluorescence associated with the Hb9::EGFP MN reporter; (C, F, I, L, O, R) red fluorescence corresponding to antibody staining for the indicated presynaptic proteins: Slc18a3 (VAChT), Slc5a7 (ChT1), Syntaxin 1a (Stx1a), Snap25, Sv2, and Synaptophysin (Syp). Scale bar is 20 µm.

    Article Snippet: The following primary antibodies were used: Slc18a3 (VAChT, Millipore; AB1588); Slc5a7 (choline transporter 1 (ChT1), Millipore; AB5966)); Sv2 (Developmental Studies Hybridoma Bank); Snap25 (Stressgen; VAP-SV0002); synaptophysin (Syp, Sigma; S-5768); syntaxin 1a (Stx1a, Stressgen; VAM-SV013).

    Techniques: Derivative Assay, In Vitro, Immunofluorescence, Fluorescence, Staining

    Differentiation Defects in Rb KO SACs (A) P18 horizontal sections of WT, Rb KO, and Rb/E2f1 DKO retina were stained for nuclei (DAPI, blue), Calb2 (green), and Slc18a3 (red). (B) Confocal images of P30 horizontal sections of WT and Rb KO retina were stained for nuclei (DAPI, blue), Chat and Slc18a3 (both red), and Camk2a (green). In the Rb KO section, the red stain is Chat only, as Slc18a3 is missing (see [A]). (C) Quantification of dense Calb2 + cell bodies in the INL, total Slc18a3 + cell bodies, and Camk2a + cell bodies in the INL. Error bars represent SD of measurements from three animals, and asterisks indicate significant differences between retinas of WT and the indicated genotypes (*, p

    Journal: PLoS Biology

    Article Title: Rb-Mediated Neuronal Differentiation through Cell-Cycle-Independent Regulation of E2f3a

    doi: 10.1371/journal.pbio.0050179

    Figure Lengend Snippet: Differentiation Defects in Rb KO SACs (A) P18 horizontal sections of WT, Rb KO, and Rb/E2f1 DKO retina were stained for nuclei (DAPI, blue), Calb2 (green), and Slc18a3 (red). (B) Confocal images of P30 horizontal sections of WT and Rb KO retina were stained for nuclei (DAPI, blue), Chat and Slc18a3 (both red), and Camk2a (green). In the Rb KO section, the red stain is Chat only, as Slc18a3 is missing (see [A]). (C) Quantification of dense Calb2 + cell bodies in the INL, total Slc18a3 + cell bodies, and Camk2a + cell bodies in the INL. Error bars represent SD of measurements from three animals, and asterisks indicate significant differences between retinas of WT and the indicated genotypes (*, p

    Article Snippet: The following primary antibodies were used: E2f-1 (SC-193), E2f-3 (SC-878), Cdkn1a (p21, SC-471), Cdkn1b (p27, SC-528), Pou4f2 (Brn3b, SC-6062), and Tfdp1 (Dp1, SC-610) from Santa Cruz Biotechnology ( http://www.scbt.com ), pRB (554136) from BD Science-Pharmingen ( http://www.bdbiosciences.com ), and Slc18a3 (VAChT, G448A) from Promega ( http://www.promega.com ).

    Techniques: Staining

    E2f3 Loss Rescues Differentiation of Rb KO SACs (A) Horizontal retinal sections of the indicated ages and genotypes were stained for nuclei (DAPI, blue), mitosis marker PH3 (green), and Slc18a3 (red), which marks SAC soma at early stages and processes from ~P5 onwards. Arrows show mitotic PH3 + nuclei in Rb KO, Rb/E2f2 DKO, and Rb/E2f3 DKO retinas. E2f1 loss rescues the ectopic mitosis and cell death defects, but not the SAC defect. E2f2 loss has no effect. E2f3 loss does not rescue the ectopic mitosis and cell loss defects, but rescues the SAC defect. Inactivating E2f1 and E2f3 together rescues the ectopic mitosis, cell death, and SAC defects. (B) The fraction of Camk2a + cells that are Chat + and Slc18a3 + in the P30 retina. (C) Horizontal P5 retinal sections were stained for nuclei (DAPI, blue), Slc18a3 (green), and Isl1 (red). Arrows show double-labelled Isl1 + /Slc18a3 + cells in the inner INL. (D) Horizontal P0 retinal sections of the indicated genotypes were stained for nuclei (DAPI, blue), cell division marker Mki67 (green), and Isl1 (red). Arrows show double-labelled dividing SACs. (E) The fraction of Isl1 + cells in the inner NBL (INBL) of P0 retinas that are dividing (Mki67 + ). Error bars represent SD of measurements from three animals, and asterisks indicate significant differences between retinas of WT and the indicated genotypes (*, p

    Journal: PLoS Biology

    Article Title: Rb-Mediated Neuronal Differentiation through Cell-Cycle-Independent Regulation of E2f3a

    doi: 10.1371/journal.pbio.0050179

    Figure Lengend Snippet: E2f3 Loss Rescues Differentiation of Rb KO SACs (A) Horizontal retinal sections of the indicated ages and genotypes were stained for nuclei (DAPI, blue), mitosis marker PH3 (green), and Slc18a3 (red), which marks SAC soma at early stages and processes from ~P5 onwards. Arrows show mitotic PH3 + nuclei in Rb KO, Rb/E2f2 DKO, and Rb/E2f3 DKO retinas. E2f1 loss rescues the ectopic mitosis and cell death defects, but not the SAC defect. E2f2 loss has no effect. E2f3 loss does not rescue the ectopic mitosis and cell loss defects, but rescues the SAC defect. Inactivating E2f1 and E2f3 together rescues the ectopic mitosis, cell death, and SAC defects. (B) The fraction of Camk2a + cells that are Chat + and Slc18a3 + in the P30 retina. (C) Horizontal P5 retinal sections were stained for nuclei (DAPI, blue), Slc18a3 (green), and Isl1 (red). Arrows show double-labelled Isl1 + /Slc18a3 + cells in the inner INL. (D) Horizontal P0 retinal sections of the indicated genotypes were stained for nuclei (DAPI, blue), cell division marker Mki67 (green), and Isl1 (red). Arrows show double-labelled dividing SACs. (E) The fraction of Isl1 + cells in the inner NBL (INBL) of P0 retinas that are dividing (Mki67 + ). Error bars represent SD of measurements from three animals, and asterisks indicate significant differences between retinas of WT and the indicated genotypes (*, p

    Article Snippet: The following primary antibodies were used: E2f-1 (SC-193), E2f-3 (SC-878), Cdkn1a (p21, SC-471), Cdkn1b (p27, SC-528), Pou4f2 (Brn3b, SC-6062), and Tfdp1 (Dp1, SC-610) from Santa Cruz Biotechnology ( http://www.scbt.com ), pRB (554136) from BD Science-Pharmingen ( http://www.bdbiosciences.com ), and Slc18a3 (VAChT, G448A) from Promega ( http://www.promega.com ).

    Techniques: Staining, Marker

    E2f3 and Rb Expression in SACs (A) Left panels: horizontal P0, P8, and P18 retinal sections of the indicated genotypes were stained for E2f3 (red) and DAPI (blue). The arrow indicates the junction between the E2f3 null peripheral and WT central P0 retina. Note the absence of E2f3 protein in the peripheral E2f3 KO RPCs at P0 and in peripheral inner retinal neurons at P18. Far right panel: P18 retinal sections of the indicated genotypes were stained for Rb (red) and DAPI (blue). Note the absence of Rb protein in the peripheral Rb KO inner retinal neurons. (B) WT P18 retinal sections were stained for nuclei (DAPI, blue), E2f3 (red) or Rb (red), and Chat plus Slc18a3 (green). Arrows indicate double-labelled soma. Note that the IPL processes are also double-labelled. Scale bars are 50 μm.

    Journal: PLoS Biology

    Article Title: Rb-Mediated Neuronal Differentiation through Cell-Cycle-Independent Regulation of E2f3a

    doi: 10.1371/journal.pbio.0050179

    Figure Lengend Snippet: E2f3 and Rb Expression in SACs (A) Left panels: horizontal P0, P8, and P18 retinal sections of the indicated genotypes were stained for E2f3 (red) and DAPI (blue). The arrow indicates the junction between the E2f3 null peripheral and WT central P0 retina. Note the absence of E2f3 protein in the peripheral E2f3 KO RPCs at P0 and in peripheral inner retinal neurons at P18. Far right panel: P18 retinal sections of the indicated genotypes were stained for Rb (red) and DAPI (blue). Note the absence of Rb protein in the peripheral Rb KO inner retinal neurons. (B) WT P18 retinal sections were stained for nuclei (DAPI, blue), E2f3 (red) or Rb (red), and Chat plus Slc18a3 (green). Arrows indicate double-labelled soma. Note that the IPL processes are also double-labelled. Scale bars are 50 μm.

    Article Snippet: The following primary antibodies were used: E2f-1 (SC-193), E2f-3 (SC-878), Cdkn1a (p21, SC-471), Cdkn1b (p27, SC-528), Pou4f2 (Brn3b, SC-6062), and Tfdp1 (Dp1, SC-610) from Santa Cruz Biotechnology ( http://www.scbt.com ), pRB (554136) from BD Science-Pharmingen ( http://www.bdbiosciences.com ), and Slc18a3 (VAChT, G448A) from Promega ( http://www.promega.com ).

    Techniques: Expressing, Staining

    Bilateral superior cervical ganglionectomy in SHR attenuates sympathetic fibre innervation of vertebrobasilar arteries but is without effect on their remodelling A , representative images of vertebrobasilar arteries showing immunofluorescence staining α‐DBH‐AF594 and α‐VAChT‐AF488 after SCGx or sham operation in SHR and corresponding bright field (BF) images. The arrowheads indicate exemplar sympathetic fibres stained with DBH antibody. B , the reduction in DBH staining is clearly evident after ganglionectomy. Percentage change in sympathetic (DBH positive) and parasympathetic (VAChT positive) fibre densities in SCGx compared to sham animals according to area. Significant differences to normalised sham values are indicated on the graph: * P

    Journal: The Journal of Physiology

    Article Title: Differences in autonomic innervation to the vertebrobasilar arteries in spontaneously hypertensive and Wistar rats

    doi: 10.1113/JP275973

    Figure Lengend Snippet: Bilateral superior cervical ganglionectomy in SHR attenuates sympathetic fibre innervation of vertebrobasilar arteries but is without effect on their remodelling A , representative images of vertebrobasilar arteries showing immunofluorescence staining α‐DBH‐AF594 and α‐VAChT‐AF488 after SCGx or sham operation in SHR and corresponding bright field (BF) images. The arrowheads indicate exemplar sympathetic fibres stained with DBH antibody. B , the reduction in DBH staining is clearly evident after ganglionectomy. Percentage change in sympathetic (DBH positive) and parasympathetic (VAChT positive) fibre densities in SCGx compared to sham animals according to area. Significant differences to normalised sham values are indicated on the graph: * P

    Article Snippet: We also emphasise immunofluorescence will not necessarily label all fibres present. α‐DBH (Millipore, Watford, UK) is specifically labelling noradrenergic fibres of the sympathetic system, whereas α‐VAChT (Millipore, Watford, UK) and α‐VIP (Novus Biologicals, Oxon, UK) stain the cholinergic and the non‐cholinergic, peptidergic component of parasympathetic fibres, respectively. α‐Neuronal nitric oxide synthase (nNOS, Santa Cruz, Insight Biotechnology, Wembley, UK) was also visualised having putative parasympathetic and/or sensory roles.

    Techniques: Immunofluorescence, Staining

    Loss of Shox2 function in the facial motor nucleus interferes with the proper development of the medial subnuclei. a Diagram of a coronal section through the P0 brain highlights the region represented in panels b – v , particularly the 7 neuronal clusters that form the facial motor nucleus ( nVII , dashed-circle ) (image adapted from [ 23 ]). b X-gal stained coronal sections through P0 brains shows a loss of medial neuronal clusters in the facial motor nucleus of Nestin - Cre; Shox2 lacZ/flox ( B ″) brains as compared to controls ( B ′). c – v ISH on P0 control ( c , e , g , i , k , m , o , q , s , u ) and Nestin - Cre; Shox2 flox/ − ( d , f , h , j , l , n , p , r , t , v ) coronal sections shows loss of Shox2 expression (compare c to d , dashed-circle ), and decreases in Isl1 (compare e to f , dashed-circle ), Phox2b (compare g to h , dashed-circle ), Cntn2 (compare i to j , dashed-circle ), Tubb3 (compare k to l , dashed-circle ), Periph (compare m to n , dashed-circle ), Slc18a3 (compare o to p , dashed-circle ), Slit2 (compare q to r , dashed-circle ), Shh (compare s to t , dashed-circle ) and Ptch1 (compare u to v , dashed-circle ) expression in the facial motor nucleus. DA dorsal accessory, DM dorsomedial, VM ventromedial, DI dorsal intermediate, VI ventral intermediate, DL dorsolateral, Lat lateral. Scale bar 250 μm.

    Journal: BMC Neuroscience

    Article Title: Shox2 is required for the proper development of the facial motor nucleus and the establishment of the facial nerves

    doi: 10.1186/s12868-015-0176-0

    Figure Lengend Snippet: Loss of Shox2 function in the facial motor nucleus interferes with the proper development of the medial subnuclei. a Diagram of a coronal section through the P0 brain highlights the region represented in panels b – v , particularly the 7 neuronal clusters that form the facial motor nucleus ( nVII , dashed-circle ) (image adapted from [ 23 ]). b X-gal stained coronal sections through P0 brains shows a loss of medial neuronal clusters in the facial motor nucleus of Nestin - Cre; Shox2 lacZ/flox ( B ″) brains as compared to controls ( B ′). c – v ISH on P0 control ( c , e , g , i , k , m , o , q , s , u ) and Nestin - Cre; Shox2 flox/ − ( d , f , h , j , l , n , p , r , t , v ) coronal sections shows loss of Shox2 expression (compare c to d , dashed-circle ), and decreases in Isl1 (compare e to f , dashed-circle ), Phox2b (compare g to h , dashed-circle ), Cntn2 (compare i to j , dashed-circle ), Tubb3 (compare k to l , dashed-circle ), Periph (compare m to n , dashed-circle ), Slc18a3 (compare o to p , dashed-circle ), Slit2 (compare q to r , dashed-circle ), Shh (compare s to t , dashed-circle ) and Ptch1 (compare u to v , dashed-circle ) expression in the facial motor nucleus. DA dorsal accessory, DM dorsomedial, VM ventromedial, DI dorsal intermediate, VI ventral intermediate, DL dorsolateral, Lat lateral. Scale bar 250 μm.

    Article Snippet: The expression levels of Isl1 , Phox2b , Cntn2 , Tubb3 , Periph , Slc18a3 and Slit2 were all strikingly reduced in the P0 facial motor nucleus of Nestin -Cre; Shox2 flox/ − mutant animals as compared to controls (Figure e–r, dashed-circle).

    Techniques: Staining, In Situ Hybridization, Expressing

    Loss of Shox2 in the brain results in subtle changes in the expression pattern of Isl1 and Phox2b in the E14.5 facial motor nucleus. a Diagram of a sagittal section through the E14.5 brain highlights the regions represented in b – r . b X-gal stained sagittal sections through E14.5 embryos shows decreased staining in the facial motor nucleus ( nVII ) of Nestin - Cre; Shox2 lacZ/flox ( B ″) embryos that lack Shox2 function, as compared to controls ( B ′). c – r ISH on E14.5 control ( c , e , g , i , k , m , o , q ) and Nestin - Cre; Shox2 flox/ − ( d , f , h , j , l , n , p , r ) sagittal sections shows loss of Shox2 expression (compare c to d , dashed-circle ), and subtle changes in the spatial expression patterns of Isl1 (compare e to f , arrow ), Phox2b (compare g to h , arrow ), Cntn2 (compare i to j , dashed-circle ), Tubb3 (compare k to l , dashed-circle ), Periph (compare m to n , arrow ), Slc18a3 (compare o to p , arrow ) and Slit2 (compare q to r , arrow ) in the developing facial motor nucleus. The dashed-circles in paired panels being compared (e.g. B ′ and B ″) are the same size, and this applies from B ′ to r . Scale bar 250 μm.

    Journal: BMC Neuroscience

    Article Title: Shox2 is required for the proper development of the facial motor nucleus and the establishment of the facial nerves

    doi: 10.1186/s12868-015-0176-0

    Figure Lengend Snippet: Loss of Shox2 in the brain results in subtle changes in the expression pattern of Isl1 and Phox2b in the E14.5 facial motor nucleus. a Diagram of a sagittal section through the E14.5 brain highlights the regions represented in b – r . b X-gal stained sagittal sections through E14.5 embryos shows decreased staining in the facial motor nucleus ( nVII ) of Nestin - Cre; Shox2 lacZ/flox ( B ″) embryos that lack Shox2 function, as compared to controls ( B ′). c – r ISH on E14.5 control ( c , e , g , i , k , m , o , q ) and Nestin - Cre; Shox2 flox/ − ( d , f , h , j , l , n , p , r ) sagittal sections shows loss of Shox2 expression (compare c to d , dashed-circle ), and subtle changes in the spatial expression patterns of Isl1 (compare e to f , arrow ), Phox2b (compare g to h , arrow ), Cntn2 (compare i to j , dashed-circle ), Tubb3 (compare k to l , dashed-circle ), Periph (compare m to n , arrow ), Slc18a3 (compare o to p , arrow ) and Slit2 (compare q to r , arrow ) in the developing facial motor nucleus. The dashed-circles in paired panels being compared (e.g. B ′ and B ″) are the same size, and this applies from B ′ to r . Scale bar 250 μm.

    Article Snippet: The expression levels of Isl1 , Phox2b , Cntn2 , Tubb3 , Periph , Slc18a3 and Slit2 were all strikingly reduced in the P0 facial motor nucleus of Nestin -Cre; Shox2 flox/ − mutant animals as compared to controls (Figure e–r, dashed-circle).

    Techniques: Expressing, Staining, In Situ Hybridization

    Shox2 is required for the proper development of the facial motor nucleus. a Diagram of a sagittal section through the P0 brain highlights the regions represented in b – v . b X-gal stained sagittal sections through P0 brains show a decrease in staining levels in the facial motor nucleus ( nVII ) of Nestin - Cre; Shox2 lacZ/flox ( B ″) animals as compared to controls ( B ′). c – v ISH on P0 control ( c , e , g , i , k , m , o , q , s , u ) and Nestin - Cre; Shox2 flox/ − ( d , f , h , j , l , n , p , r , t , v ) sagittal sections shows loss of Shox2 expression (compare c to d , dashed-circle ), and decreases in Isl1 (compare e to f , dashed-circle ), Phox2b (compare g to h , dashed-circle ), Cntn2 (compare i to j , dashed-circle ), Tubb3 (compare k to l , dashed-circle ), Periph (compare m to n , dashed-circle ), Slc18a3 (compare o to p , dashed-circle ), Slit2 (compare q to r , dashed-circle ), Shh (compare s to t , dashed-circle ) and Ptch1 (compare u to v , dashed-circle ) expression in the facial motor nucleus. nV trigeminal nucleus, RTN retrotrapezoid nucleus. Scale bar 250 μm.

    Journal: BMC Neuroscience

    Article Title: Shox2 is required for the proper development of the facial motor nucleus and the establishment of the facial nerves

    doi: 10.1186/s12868-015-0176-0

    Figure Lengend Snippet: Shox2 is required for the proper development of the facial motor nucleus. a Diagram of a sagittal section through the P0 brain highlights the regions represented in b – v . b X-gal stained sagittal sections through P0 brains show a decrease in staining levels in the facial motor nucleus ( nVII ) of Nestin - Cre; Shox2 lacZ/flox ( B ″) animals as compared to controls ( B ′). c – v ISH on P0 control ( c , e , g , i , k , m , o , q , s , u ) and Nestin - Cre; Shox2 flox/ − ( d , f , h , j , l , n , p , r , t , v ) sagittal sections shows loss of Shox2 expression (compare c to d , dashed-circle ), and decreases in Isl1 (compare e to f , dashed-circle ), Phox2b (compare g to h , dashed-circle ), Cntn2 (compare i to j , dashed-circle ), Tubb3 (compare k to l , dashed-circle ), Periph (compare m to n , dashed-circle ), Slc18a3 (compare o to p , dashed-circle ), Slit2 (compare q to r , dashed-circle ), Shh (compare s to t , dashed-circle ) and Ptch1 (compare u to v , dashed-circle ) expression in the facial motor nucleus. nV trigeminal nucleus, RTN retrotrapezoid nucleus. Scale bar 250 μm.

    Article Snippet: The expression levels of Isl1 , Phox2b , Cntn2 , Tubb3 , Periph , Slc18a3 and Slit2 were all strikingly reduced in the P0 facial motor nucleus of Nestin -Cre; Shox2 flox/ − mutant animals as compared to controls (Figure e–r, dashed-circle).

    Techniques: Staining, In Situ Hybridization, Expressing

    ChIP-seq assays revealed Isl-Lhx3-hexamer-binding sites in a cholinergic gene battery. ( A ) Schematic representation of cholinergic neurotransmission system. Acly, ATP-citrate lyase; CoA, coenzyme A; ChAT, choline acetyltransferase; ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; AChE, Acetylcholine esterase; CHT, high affinity choline transporter; AChRs, Acetylcholine receptors. ( B ) ChIP-seq tag profile of the genomic region surrounding a battery of cholinergic genes ChAT/VAChT , CHT , and Acly loci. Each cholinergic gene is indicated, and the blue arrows represent the direction of transcription. Mam cons., mammalian conservation. The ChIP-seq data was deposited in the GEO database (assession no. GSE50993) [20] . ( C ) Schematic representation of the location of the HxRE motifs in each of the 500 bp-long cholinergic gene peaks. The number shows the relative position within the peak (0, the center position of each peak). ( D ) In vivo ChIP assays in dissected E12.5 embryonic spinal cords to monitor the binding of the Isl1-Lhx3-hexamer to the cholinergic enhancers. Schematic representation of the ChAT gene is shown on the top. The arrows indicate two sets of primers detecting ChAT -enhancer ( ChAT -enh) and a negative control region lacking the Isl1-Lhx3-binding peak ( ChAT -neg). Isl1, Lhx3, and NLI were recruited to the cholinergic enhancers in embryonic spinal cords. Error bars indicate standard deviation.

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: ChIP-seq assays revealed Isl-Lhx3-hexamer-binding sites in a cholinergic gene battery. ( A ) Schematic representation of cholinergic neurotransmission system. Acly, ATP-citrate lyase; CoA, coenzyme A; ChAT, choline acetyltransferase; ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; AChE, Acetylcholine esterase; CHT, high affinity choline transporter; AChRs, Acetylcholine receptors. ( B ) ChIP-seq tag profile of the genomic region surrounding a battery of cholinergic genes ChAT/VAChT , CHT , and Acly loci. Each cholinergic gene is indicated, and the blue arrows represent the direction of transcription. Mam cons., mammalian conservation. The ChIP-seq data was deposited in the GEO database (assession no. GSE50993) [20] . ( C ) Schematic representation of the location of the HxRE motifs in each of the 500 bp-long cholinergic gene peaks. The number shows the relative position within the peak (0, the center position of each peak). ( D ) In vivo ChIP assays in dissected E12.5 embryonic spinal cords to monitor the binding of the Isl1-Lhx3-hexamer to the cholinergic enhancers. Schematic representation of the ChAT gene is shown on the top. The arrows indicate two sets of primers detecting ChAT -enhancer ( ChAT -enh) and a negative control region lacking the Isl1-Lhx3-binding peak ( ChAT -neg). Isl1, Lhx3, and NLI were recruited to the cholinergic enhancers in embryonic spinal cords. Error bars indicate standard deviation.

    Article Snippet: For quantitative PCR of ChAT, VAChT, Acly and CHT, the following probes and primers predesigned by the TaqMan Gene Expression Assay (Applied Biosystems) for each gene were used with TaqMan Universial Master MixII and 7500 ABI qPCR machine (Applied Biosystems); ChAT (Assay ID-Mm01221882_m1), VAChT (Assay ID-Mm00491465_s1), Acly (Assay ID-Mm01302282_m1), CHT (Assay ID- Mm00452075_m1) and Eukaryotic 18S rRNA Endogenous Control (FAM Dye/MGB Probe, Non-Primer Limited).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Negative Control, Standard Deviation

    Isl1 is required for the formation of striatal cholinergic interneurons in the developing forebrain. Immunohistochemical analyses on the CPu (A, B) and BMC (C, D) of E16.5 Isl1 f/f ;Nkx2.1Cre and littermate control embryos. VAChT + cholinergic interneurons in the CPu failed to form in the MGE-specific Isl1 -null embryos. (E, F) Quantification of the number of Lhx8 + VAChT + (E) or Nkx2.1 + VAChT + cells in the CPu of E16.5 control and Isl1 mutant embryos. Histogram shows average ± standard deviation. ** p

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Isl1 is required for the formation of striatal cholinergic interneurons in the developing forebrain. Immunohistochemical analyses on the CPu (A, B) and BMC (C, D) of E16.5 Isl1 f/f ;Nkx2.1Cre and littermate control embryos. VAChT + cholinergic interneurons in the CPu failed to form in the MGE-specific Isl1 -null embryos. (E, F) Quantification of the number of Lhx8 + VAChT + (E) or Nkx2.1 + VAChT + cells in the CPu of E16.5 control and Isl1 mutant embryos. Histogram shows average ± standard deviation. ** p

    Article Snippet: For quantitative PCR of ChAT, VAChT, Acly and CHT, the following probes and primers predesigned by the TaqMan Gene Expression Assay (Applied Biosystems) for each gene were used with TaqMan Universial Master MixII and 7500 ABI qPCR machine (Applied Biosystems); ChAT (Assay ID-Mm01221882_m1), VAChT (Assay ID-Mm00491465_s1), Acly (Assay ID-Mm01302282_m1), CHT (Assay ID- Mm00452075_m1) and Eukaryotic 18S rRNA Endogenous Control (FAM Dye/MGB Probe, Non-Primer Limited).

    Techniques: Immunohistochemistry, Mutagenesis, Standard Deviation

    Isl1-Lhx8 induces a cholinergic fate in ESC-derived neurons. ( A, B ) In Isl1-Lhx8-ESCs, the expression of Flag-tagged Isl1-Lhx8 was induced by Dox, as detected by western blotting assays with α-Flag antibodies (A) and immunohistochemistry assays with α-Isl1 and α-Lhx8 antibodies (B). ( C, D ) Quantitative RT-PCR analyses in Isl1-Lhx8-ESCs when cultured as a monolayer. Cholinergic genes, but not the MN gene Hb9, were induced by Isl1-Lhx8. ( E–G ) Cell differentiation analyses in floating EBs derived from Isl1-Lhx8-ESCs, cultured with or without Dox, which triggers expression of Isl1-Lhx8. Immunohistochemical analyses show that Isl-Lhx8 expression induces differentiation of VAChT + TuJ + c holinergic neurons (E, F). Quantitative RT-PCR analyses show that cholinergic pathway genes, but not MN genes Hb9 and Isl2, were induced by Isl1-Lhx8 (G). ( H ) Quantitative RT-PCR analyses of Hb9 expression in Isl1-Lhx3-ESCs. Hb9 was induced by Dox treatment, which induces the expression of Isl1-Lhx3 in Isl1-Lhx3-ESCs, when cultured in either monolayer (M) or spinal neuronal differentiation (SN) conditions. Error bars represent the standard deviation in all graphs (C, D, G, H).

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Isl1-Lhx8 induces a cholinergic fate in ESC-derived neurons. ( A, B ) In Isl1-Lhx8-ESCs, the expression of Flag-tagged Isl1-Lhx8 was induced by Dox, as detected by western blotting assays with α-Flag antibodies (A) and immunohistochemistry assays with α-Isl1 and α-Lhx8 antibodies (B). ( C, D ) Quantitative RT-PCR analyses in Isl1-Lhx8-ESCs when cultured as a monolayer. Cholinergic genes, but not the MN gene Hb9, were induced by Isl1-Lhx8. ( E–G ) Cell differentiation analyses in floating EBs derived from Isl1-Lhx8-ESCs, cultured with or without Dox, which triggers expression of Isl1-Lhx8. Immunohistochemical analyses show that Isl-Lhx8 expression induces differentiation of VAChT + TuJ + c holinergic neurons (E, F). Quantitative RT-PCR analyses show that cholinergic pathway genes, but not MN genes Hb9 and Isl2, were induced by Isl1-Lhx8 (G). ( H ) Quantitative RT-PCR analyses of Hb9 expression in Isl1-Lhx3-ESCs. Hb9 was induced by Dox treatment, which induces the expression of Isl1-Lhx3 in Isl1-Lhx3-ESCs, when cultured in either monolayer (M) or spinal neuronal differentiation (SN) conditions. Error bars represent the standard deviation in all graphs (C, D, G, H).

    Article Snippet: For quantitative PCR of ChAT, VAChT, Acly and CHT, the following probes and primers predesigned by the TaqMan Gene Expression Assay (Applied Biosystems) for each gene were used with TaqMan Universial Master MixII and 7500 ABI qPCR machine (Applied Biosystems); ChAT (Assay ID-Mm01221882_m1), VAChT (Assay ID-Mm00491465_s1), Acly (Assay ID-Mm01302282_m1), CHT (Assay ID- Mm00452075_m1) and Eukaryotic 18S rRNA Endogenous Control (FAM Dye/MGB Probe, Non-Primer Limited).

    Techniques: Derivative Assay, Expressing, Western Blot, Immunohistochemistry, Quantitative RT-PCR, Cell Culture, Cell Differentiation, Standard Deviation

    Isl1-Lhx8 induces the expression of cholinergic gene battery in the forebrain, but not in the spinal cord. ( A ) Schematic representation of in utero electroporation of the cortex, followed by quantitative RT-PCR (qRT-PCR) analyses. E13.5 brains were subjected to electroporation after each combination of constructs was injected into the lateral ventricle. The GFP + region of electroporated (+) cortex and the comparable region of unelectroporated (−) cortex were micro-dissected and analyzed for gene expression. ( B ) Expression analyses of the cholinergic pathway genes, ChAT, VAChT and CHT , in mouse cortices electroporated with constructs, as indicated by color bars. Y-axis indicates the relative expression levels of each cholinergic gene on the electropoated side over the control side. The expression of Isl1-Lhx8 led to upregulation of the cholinergic genes in the cortex, whereas that of Isl1-Lhx3 failed to induce cholinergic genes in this context. Error bars indicate standard deviation. ** p

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Isl1-Lhx8 induces the expression of cholinergic gene battery in the forebrain, but not in the spinal cord. ( A ) Schematic representation of in utero electroporation of the cortex, followed by quantitative RT-PCR (qRT-PCR) analyses. E13.5 brains were subjected to electroporation after each combination of constructs was injected into the lateral ventricle. The GFP + region of electroporated (+) cortex and the comparable region of unelectroporated (−) cortex were micro-dissected and analyzed for gene expression. ( B ) Expression analyses of the cholinergic pathway genes, ChAT, VAChT and CHT , in mouse cortices electroporated with constructs, as indicated by color bars. Y-axis indicates the relative expression levels of each cholinergic gene on the electropoated side over the control side. The expression of Isl1-Lhx8 led to upregulation of the cholinergic genes in the cortex, whereas that of Isl1-Lhx3 failed to induce cholinergic genes in this context. Error bars indicate standard deviation. ** p

    Article Snippet: For quantitative PCR of ChAT, VAChT, Acly and CHT, the following probes and primers predesigned by the TaqMan Gene Expression Assay (Applied Biosystems) for each gene were used with TaqMan Universial Master MixII and 7500 ABI qPCR machine (Applied Biosystems); ChAT (Assay ID-Mm01221882_m1), VAChT (Assay ID-Mm00491465_s1), Acly (Assay ID-Mm01302282_m1), CHT (Assay ID- Mm00452075_m1) and Eukaryotic 18S rRNA Endogenous Control (FAM Dye/MGB Probe, Non-Primer Limited).

    Techniques: Expressing, In Utero, Electroporation, Quantitative RT-PCR, Construct, Injection, Standard Deviation

    The Isl1-Lhx3-hexamer plays a crucial role in inducing the expression of cholinergic pathway genes in the developing spinal MNs. ( A ) Expression analyses of the cholinergic pathway genes in chick embryos electroporated with Isl1 and Lhx3 using in situ hybridization. The co-electroporation of Isl1 and Lhx3 triggered the ectopic expression of cholinergic genes in the dorsal spinal cord, as marked by brackets. + indicates the electroporated side. ( B ) Expression analyses of the cholinergic pathway genes using either immunohistochemistry or in situ hybridization on the spinal cord of E12.5 Isl1 f/f ;nestinCre and littermate control embryos. The ventral quadrant spinal cord is shown. The cholinergic genes are markedly downregulated in Isl1 f/f ;nestinCre mice. The remaining VAChT expression is correlated with the residual Isl1 expression in Isl1 f/f ;nestinCre mice, as determined by immunostaining assays.

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: The Isl1-Lhx3-hexamer plays a crucial role in inducing the expression of cholinergic pathway genes in the developing spinal MNs. ( A ) Expression analyses of the cholinergic pathway genes in chick embryos electroporated with Isl1 and Lhx3 using in situ hybridization. The co-electroporation of Isl1 and Lhx3 triggered the ectopic expression of cholinergic genes in the dorsal spinal cord, as marked by brackets. + indicates the electroporated side. ( B ) Expression analyses of the cholinergic pathway genes using either immunohistochemistry or in situ hybridization on the spinal cord of E12.5 Isl1 f/f ;nestinCre and littermate control embryos. The ventral quadrant spinal cord is shown. The cholinergic genes are markedly downregulated in Isl1 f/f ;nestinCre mice. The remaining VAChT expression is correlated with the residual Isl1 expression in Isl1 f/f ;nestinCre mice, as determined by immunostaining assays.

    Article Snippet: For quantitative PCR of ChAT, VAChT, Acly and CHT, the following probes and primers predesigned by the TaqMan Gene Expression Assay (Applied Biosystems) for each gene were used with TaqMan Universial Master MixII and 7500 ABI qPCR machine (Applied Biosystems); ChAT (Assay ID-Mm01221882_m1), VAChT (Assay ID-Mm00491465_s1), Acly (Assay ID-Mm01302282_m1), CHT (Assay ID- Mm00452075_m1) and Eukaryotic 18S rRNA Endogenous Control (FAM Dye/MGB Probe, Non-Primer Limited).

    Techniques: Expressing, In Situ Hybridization, Electroporation, Immunohistochemistry, Mouse Assay, Immunostaining

    Human detrusor ICCs. A , human detrusor labeled with anti-SM myosin ( SMM ). B , at mucosa-detrusor interface SMCs were arranged in loose network, which was organized into distinct bundles in detrusor muscularis. C to E , representative human detrusor co-labeled with anti-SM myosin and anti- KIT (green areas). Elongated, branched KIT positive ICC track SM bundles and occupy spaces between bundles with obvious spiky morphology. E , nuclei counterstained with 4,6-diamidino-2-phenylindole (blue areas). F , detrusor ICCs (green areas) associated with vAChT nerves (red areas).

    Journal: The Journal of Urology

    Article Title: Morphological Expression of KIT Positive Interstitial Cells of Cajal in Human Bladder

    doi: 10.1016/j.juro.2010.03.005

    Figure Lengend Snippet: Human detrusor ICCs. A , human detrusor labeled with anti-SM myosin ( SMM ). B , at mucosa-detrusor interface SMCs were arranged in loose network, which was organized into distinct bundles in detrusor muscularis. C to E , representative human detrusor co-labeled with anti-SM myosin and anti- KIT (green areas). Elongated, branched KIT positive ICC track SM bundles and occupy spaces between bundles with obvious spiky morphology. E , nuclei counterstained with 4,6-diamidino-2-phenylindole (blue areas). F , detrusor ICCs (green areas) associated with vAChT nerves (red areas).

    Article Snippet: Primary antibodies were raised against human immunogens, including anti-KIT (1:200), anti-vimentin (1:200), anti-SM myosin (1:200) (Sigma®), anti-vAChT (Santa Cruz Biotechnology, Santa Cruz, California) (1:2,000) and anti-mast cell tryptase (Abcam®) (1:200).

    Techniques: Labeling, Immunocytochemistry

    ICCs and mucosal cholinergic nerves. A , cholinergic nerve plexus in human bladder mucosa labeled with anti-vAChT. B and C , low magnification of preparations co-labeled with anti- KIT (green areas) and anti-vAChT (red areas) show ICC network-cholinergic plexus associations. D to F , high magnification reveals ICCs and cholinergic nerves with points of close association (arrows).

    Journal: The Journal of Urology

    Article Title: Morphological Expression of KIT Positive Interstitial Cells of Cajal in Human Bladder

    doi: 10.1016/j.juro.2010.03.005

    Figure Lengend Snippet: ICCs and mucosal cholinergic nerves. A , cholinergic nerve plexus in human bladder mucosa labeled with anti-vAChT. B and C , low magnification of preparations co-labeled with anti- KIT (green areas) and anti-vAChT (red areas) show ICC network-cholinergic plexus associations. D to F , high magnification reveals ICCs and cholinergic nerves with points of close association (arrows).

    Article Snippet: Primary antibodies were raised against human immunogens, including anti-KIT (1:200), anti-vimentin (1:200), anti-SM myosin (1:200) (Sigma®), anti-vAChT (Santa Cruz Biotechnology, Santa Cruz, California) (1:2,000) and anti-mast cell tryptase (Abcam®) (1:200).

    Techniques: Labeling, Immunocytochemistry

    Sensory neurites sprouting in bladder wall in CYPc rats. A , Nerve fibers stained for PGP9.5 and Tuj1 in whole mount urothelium - scale bar : 50 µm. B, Fibers are stained by CGRP antibody but not by VAChT antibody in control condition. C, PGP9.5 staining of whole mount bladder mucosa in control and CYP-treated rats. D, Line representation showing neurites based on the images in panel C , illustrating increased outgrowth in CYP c . E-G, Statistical comparison of neurite segments ( E ), neurite length ( F ) and neurite density ( G ) between control and CYP c rats. H , Neurite fibers observed in CYP c are stained by CGRP but by VAChT antibody.

    Journal: PLoS ONE

    Article Title: Crucial Role of TRPC1 and TRPC4 in Cystitis-Induced Neuronal Sprouting and Bladder Overactivity

    doi: 10.1371/journal.pone.0069550

    Figure Lengend Snippet: Sensory neurites sprouting in bladder wall in CYPc rats. A , Nerve fibers stained for PGP9.5 and Tuj1 in whole mount urothelium - scale bar : 50 µm. B, Fibers are stained by CGRP antibody but not by VAChT antibody in control condition. C, PGP9.5 staining of whole mount bladder mucosa in control and CYP-treated rats. D, Line representation showing neurites based on the images in panel C , illustrating increased outgrowth in CYP c . E-G, Statistical comparison of neurite segments ( E ), neurite length ( F ) and neurite density ( G ) between control and CYP c rats. H , Neurite fibers observed in CYP c are stained by CGRP but by VAChT antibody.

    Article Snippet: The mucosa was gently harvested from the body of the bladder with forceps (excluding muscle fibers from the preparation), washed in PBS and incubated sequentially for 2 h with PBT0.3% +20% serum, overnight at 4°C with primary antibodies (PGP9.5 (Millipore - 1∶500), Tuj1 (Millipore - 1∶1000), VAChT (Santa Cruz –1∶500) and CGRP (Sigma - 1∶500)) in PBT0.3% +5% serum and for 2 h at room temperature with secondary antibodies at 1∶500.

    Techniques: Staining

    Effects of CHAT knockdown in medial habenula on depression-like behaviors. ( a ) Schematic representation of the AAV vector engineered to induce CHAT knockdown. ( b ) Experimental paradigm for behavioral testing of rats infected with the virus. ( c ) Representative photomicrographs of Hb slices from the rats injected with AAV-sh-SCR (control) or AAV-sh-CHAT (CHAT knockdown). CHAT protein expression was visualized as red immunofluorescence. Cell nuclei and viral infection were visualized using Hoechst staining (blue) and GFP (green), respectively. The inset box shows a higher magnification of an infected MHb region. None of the neurons infected with the AAV-sh-CHAT showed CHAT expression. Scale bar, 50 μm. ( d ) VACHT expression is not affected by AAV-sh-CHAT. Hb neurons injected with AAV-sh-CHAT were immunostained for VACHT (red) and EGFP (green). Scale bar, 50 μm. ( e ) CHAT and VACHT western blots from the Hb of rats injected with AAV-sh-CHAT. Rats were injected with AAV-sh-SCR (control) or AAV-sh-CHAT, which were expressed for 3 weeks before western blot analysis. ( f ) Quantification of the effects of shRNA-mediated CHAT knockdown. Data represent mean ± SEM (sh-SCR, n = 4 rats; sh-CHAT, n = 4; * P

    Journal: Scientific Reports

    Article Title: Down-regulation of cholinergic signaling in the habenula induces anhedonia-like behavior

    doi: 10.1038/s41598-017-01088-6

    Figure Lengend Snippet: Effects of CHAT knockdown in medial habenula on depression-like behaviors. ( a ) Schematic representation of the AAV vector engineered to induce CHAT knockdown. ( b ) Experimental paradigm for behavioral testing of rats infected with the virus. ( c ) Representative photomicrographs of Hb slices from the rats injected with AAV-sh-SCR (control) or AAV-sh-CHAT (CHAT knockdown). CHAT protein expression was visualized as red immunofluorescence. Cell nuclei and viral infection were visualized using Hoechst staining (blue) and GFP (green), respectively. The inset box shows a higher magnification of an infected MHb region. None of the neurons infected with the AAV-sh-CHAT showed CHAT expression. Scale bar, 50 μm. ( d ) VACHT expression is not affected by AAV-sh-CHAT. Hb neurons injected with AAV-sh-CHAT were immunostained for VACHT (red) and EGFP (green). Scale bar, 50 μm. ( e ) CHAT and VACHT western blots from the Hb of rats injected with AAV-sh-CHAT. Rats were injected with AAV-sh-SCR (control) or AAV-sh-CHAT, which were expressed for 3 weeks before western blot analysis. ( f ) Quantification of the effects of shRNA-mediated CHAT knockdown. Data represent mean ± SEM (sh-SCR, n = 4 rats; sh-CHAT, n = 4; * P

    Article Snippet: Immunohistochemistry and Western Blot For verification of AAV-mediated CHAT silencing in habenula, coronal rat brain sections (30 μm) permeabilized by 0.3% Triton X-100 in PBS were incubated with primary antibodies against CHAT (1:100, Abcam), EGFP (1:500, Abcam), VACHT (1:500, Synaptic Systems), and 5-HT (1:2000, ImmunoStar) followed by Cy3- and Alexa 488-conjugated secondary antibodies (Jackson ImmunoResearch).

    Techniques: Plasmid Preparation, Infection, Injection, Expressing, Immunofluorescence, Staining, Western Blot, shRNA

    Effect of chronic restraint stress on the expression of cholinergic system genes in the rat habenula. ( a ) Schematic diagram of a generalized cholinergic synapse. The choline transporter protein (CHT) delivers choline into the cytoplasm, where choline acetyltransferase (CHAT) catalyze the transfer of an acetyl group from the coenzyme, acetyl Co-A, to choline, generating acetylcholine, and the vesicular acetylcholine transporter (VACHT) packs acetylcholine into the vesicle. Presynaptic nicotinic acetylcholine receptors (nAChR; CHRNA3, CHRNB3 and CHRNB4) modulate the release of neurotransmitter. ( b – h ) qRT-PCR was used to quantify mRNA expression levels of six cholinergic system genes in the habenula of rats exposed to CRS. The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were significantly reduced by CRS. Values for each individual gene were normalized to the mean of the reference gene GAPDH. CAMK2B was not changed by CRS. Data represent mean ± SEM. NS, n = 4; CRS, n = 4; * P

    Journal: Scientific Reports

    Article Title: Down-regulation of cholinergic signaling in the habenula induces anhedonia-like behavior

    doi: 10.1038/s41598-017-01088-6

    Figure Lengend Snippet: Effect of chronic restraint stress on the expression of cholinergic system genes in the rat habenula. ( a ) Schematic diagram of a generalized cholinergic synapse. The choline transporter protein (CHT) delivers choline into the cytoplasm, where choline acetyltransferase (CHAT) catalyze the transfer of an acetyl group from the coenzyme, acetyl Co-A, to choline, generating acetylcholine, and the vesicular acetylcholine transporter (VACHT) packs acetylcholine into the vesicle. Presynaptic nicotinic acetylcholine receptors (nAChR; CHRNA3, CHRNB3 and CHRNB4) modulate the release of neurotransmitter. ( b – h ) qRT-PCR was used to quantify mRNA expression levels of six cholinergic system genes in the habenula of rats exposed to CRS. The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were significantly reduced by CRS. Values for each individual gene were normalized to the mean of the reference gene GAPDH. CAMK2B was not changed by CRS. Data represent mean ± SEM. NS, n = 4; CRS, n = 4; * P

    Article Snippet: Immunohistochemistry and Western Blot For verification of AAV-mediated CHAT silencing in habenula, coronal rat brain sections (30 μm) permeabilized by 0.3% Triton X-100 in PBS were incubated with primary antibodies against CHAT (1:100, Abcam), EGFP (1:500, Abcam), VACHT (1:500, Synaptic Systems), and 5-HT (1:2000, ImmunoStar) followed by Cy3- and Alexa 488-conjugated secondary antibodies (Jackson ImmunoResearch).

    Techniques: Expressing, Quantitative RT-PCR

    Expression of cholinergic system genes in habenula tissue obtained from persons with MDD who died by suicide. ( a ) The human brain was separated into two hemispheres by slicing through the center of the brain stem and cerebellum. Each hemisphere was sliced coronally into 18–20 slabs (slab 1, frontal lobe; slab 18, occipital lobe) and these slabs were frozen. The habenula is nearly adjacent to the pineal gland and is contained in the 11th of 18 slabs. To isolate habenula samples, a carving tool with a round tip was used to make long grooves one at a time. ( c – i ) The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were evaluated using TaqMan probe-based qRT-PCR. All cholinergic system genes examined were down-regulated in suicide, but not the control CAMK2B gene ( b ), an excitatory neuronal marker. GAPDH and β-Actin were used as reference genes to normalize qRT-PCR data. Data represent mean ± SEM (CON, Control, n = 11; MDD, n = 12 subjects; * P

    Journal: Scientific Reports

    Article Title: Down-regulation of cholinergic signaling in the habenula induces anhedonia-like behavior

    doi: 10.1038/s41598-017-01088-6

    Figure Lengend Snippet: Expression of cholinergic system genes in habenula tissue obtained from persons with MDD who died by suicide. ( a ) The human brain was separated into two hemispheres by slicing through the center of the brain stem and cerebellum. Each hemisphere was sliced coronally into 18–20 slabs (slab 1, frontal lobe; slab 18, occipital lobe) and these slabs were frozen. The habenula is nearly adjacent to the pineal gland and is contained in the 11th of 18 slabs. To isolate habenula samples, a carving tool with a round tip was used to make long grooves one at a time. ( c – i ) The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were evaluated using TaqMan probe-based qRT-PCR. All cholinergic system genes examined were down-regulated in suicide, but not the control CAMK2B gene ( b ), an excitatory neuronal marker. GAPDH and β-Actin were used as reference genes to normalize qRT-PCR data. Data represent mean ± SEM (CON, Control, n = 11; MDD, n = 12 subjects; * P

    Article Snippet: Immunohistochemistry and Western Blot For verification of AAV-mediated CHAT silencing in habenula, coronal rat brain sections (30 μm) permeabilized by 0.3% Triton X-100 in PBS were incubated with primary antibodies against CHAT (1:100, Abcam), EGFP (1:500, Abcam), VACHT (1:500, Synaptic Systems), and 5-HT (1:2000, ImmunoStar) followed by Cy3- and Alexa 488-conjugated secondary antibodies (Jackson ImmunoResearch).

    Techniques: Expressing, Quantitative RT-PCR, Marker

    Genetic rescue of VAChT-mutant mice. a) VAChT, ChAT, and CHT1 mRNA levels were measured by qPCR in the striatum of WT mice (white bar), VAChT WT/Flox (grey bar) and VAChT Flox/Flox (black bar) mice. b) VAChT, ChAT, CHT1 mRNA levels in the spinal cord of WT mice (white bar), VAChT WT/Flox (grey bar) and VAChT Flox/Flox (black bar) mice. c) Representative immunoblot of control, VAChT WT/Flox and VAChT Flox/Flox mice in striatum. d) Quantification of protein levels. Actin immunoreactivity was used to correct for protein loading between experiments. Data are presented as a percentage of wild-type levels. e) Representative immunoblot of control, VAChT WT/Flox and VAChT Flox/Flox mice in spinal cord. f) Quantification of protein levels. Actin immunoreactivity was used to correct for protein loading between experiments. Data are presented as a percentage of wild-type levels.

    Journal: PLoS ONE

    Article Title: Novel Strains of Mice Deficient for the Vesicular Acetylcholine Transporter: Insights on Transcriptional Regulation and Control of Locomotor Behavior

    doi: 10.1371/journal.pone.0017611

    Figure Lengend Snippet: Genetic rescue of VAChT-mutant mice. a) VAChT, ChAT, and CHT1 mRNA levels were measured by qPCR in the striatum of WT mice (white bar), VAChT WT/Flox (grey bar) and VAChT Flox/Flox (black bar) mice. b) VAChT, ChAT, CHT1 mRNA levels in the spinal cord of WT mice (white bar), VAChT WT/Flox (grey bar) and VAChT Flox/Flox (black bar) mice. c) Representative immunoblot of control, VAChT WT/Flox and VAChT Flox/Flox mice in striatum. d) Quantification of protein levels. Actin immunoreactivity was used to correct for protein loading between experiments. Data are presented as a percentage of wild-type levels. e) Representative immunoblot of control, VAChT WT/Flox and VAChT Flox/Flox mice in spinal cord. f) Quantification of protein levels. Actin immunoreactivity was used to correct for protein loading between experiments. Data are presented as a percentage of wild-type levels.

    Article Snippet: Antibodies used were anti-VAChT (rabbit polyclonal 1∶2000, Synaptic System, Germany), anti-CHT1 (rabbit polyclonal 1∶1000, kindly provided by R. Jane Rylett, University of Western Ontario, London, Canada), anti-CHAT (rabbit polyclonal 1∶1000, Chemicon) and anti-actin (Chemicon, CA).

    Techniques: Mutagenesis, Mouse Assay, Real-time Polymerase Chain Reaction

    VAChT immunorreactivity is altered in VAChT mutant mice. a) Representative optical sections from striatum stained with a VAChT antibody (green) or b) stained with CHT1 antibody (green). c) Representative optical sections from hippocampus stained with a VAChT antibody or d) CHT1 antibody (green). Dapi labelling (blue) was used to stain nuclei. Scale bar 50 µm.

    Journal: PLoS ONE

    Article Title: Novel Strains of Mice Deficient for the Vesicular Acetylcholine Transporter: Insights on Transcriptional Regulation and Control of Locomotor Behavior

    doi: 10.1371/journal.pone.0017611

    Figure Lengend Snippet: VAChT immunorreactivity is altered in VAChT mutant mice. a) Representative optical sections from striatum stained with a VAChT antibody (green) or b) stained with CHT1 antibody (green). c) Representative optical sections from hippocampus stained with a VAChT antibody or d) CHT1 antibody (green). Dapi labelling (blue) was used to stain nuclei. Scale bar 50 µm.

    Article Snippet: Antibodies used were anti-VAChT (rabbit polyclonal 1∶2000, Synaptic System, Germany), anti-CHT1 (rabbit polyclonal 1∶1000, kindly provided by R. Jane Rylett, University of Western Ontario, London, Canada), anti-CHAT (rabbit polyclonal 1∶1000, Chemicon) and anti-actin (Chemicon, CA).

    Techniques: Mutagenesis, Mouse Assay, Staining

    VAChT immunorreactivity is altered in VAChT mutant mice. a) Representative optical sections from cortex stained with a VAChT antibody (green) or b) CHT1 antibody (green). c) Representative optical sections from facial motor nuclei stained with a VAChT antibody or d) CHT1 antibody (green). Dapi labelling (blue) was used to stain nuclei. Scale bar 50 µm.

    Journal: PLoS ONE

    Article Title: Novel Strains of Mice Deficient for the Vesicular Acetylcholine Transporter: Insights on Transcriptional Regulation and Control of Locomotor Behavior

    doi: 10.1371/journal.pone.0017611

    Figure Lengend Snippet: VAChT immunorreactivity is altered in VAChT mutant mice. a) Representative optical sections from cortex stained with a VAChT antibody (green) or b) CHT1 antibody (green). c) Representative optical sections from facial motor nuclei stained with a VAChT antibody or d) CHT1 antibody (green). Dapi labelling (blue) was used to stain nuclei. Scale bar 50 µm.

    Article Snippet: Antibodies used were anti-VAChT (rabbit polyclonal 1∶2000, Synaptic System, Germany), anti-CHT1 (rabbit polyclonal 1∶1000, kindly provided by R. Jane Rylett, University of Western Ontario, London, Canada), anti-CHAT (rabbit polyclonal 1∶1000, Chemicon) and anti-actin (Chemicon, CA).

    Techniques: Mutagenesis, Mouse Assay, Staining

    VAChT mRNA expression is changed in VAChT mutant mice. a) VAChT, ChAT, CHT1 and AChase mRNA levels in striatum and b) spinal cord of WT and VAChT FloxNeo/FloxNeo mice. c) VAChT, ChAT, CHT1 and AChase mRNA levels in striatum and d) spinal cord of VAChT WT/WT , VAChT FloxNeo/Del and VAChT WT/Del mice. mRNA expression levels were quantified by qPCR using actin to normalize the data. Graphs represent average of 4–6 different mice. (*) and (**) indicate p

    Journal: PLoS ONE

    Article Title: Novel Strains of Mice Deficient for the Vesicular Acetylcholine Transporter: Insights on Transcriptional Regulation and Control of Locomotor Behavior

    doi: 10.1371/journal.pone.0017611

    Figure Lengend Snippet: VAChT mRNA expression is changed in VAChT mutant mice. a) VAChT, ChAT, CHT1 and AChase mRNA levels in striatum and b) spinal cord of WT and VAChT FloxNeo/FloxNeo mice. c) VAChT, ChAT, CHT1 and AChase mRNA levels in striatum and d) spinal cord of VAChT WT/WT , VAChT FloxNeo/Del and VAChT WT/Del mice. mRNA expression levels were quantified by qPCR using actin to normalize the data. Graphs represent average of 4–6 different mice. (*) and (**) indicate p

    Article Snippet: Antibodies used were anti-VAChT (rabbit polyclonal 1∶2000, Synaptic System, Germany), anti-CHT1 (rabbit polyclonal 1∶1000, kindly provided by R. Jane Rylett, University of Western Ontario, London, Canada), anti-CHAT (rabbit polyclonal 1∶1000, Chemicon) and anti-actin (Chemicon, CA).

    Techniques: Expressing, Mutagenesis, Mouse Assay, Real-time Polymerase Chain Reaction

    Molecular distinctions among αRGCs. A, B : Retinal whole mounts were stained with antibodies to Opn (green), plus antibodies to one of 3 POU-domain transcription factors (Brn3a, Brn3b or Brn3c; red in A ) or one of 3 calcium binding proteins (parvalbumin [PV], calbindin or calretinin; red in B ). Brn3b and PV mark most αRGCs whereas Brn3a, Brn3c and calbindin mark subsets; most αRGCs are calretinin-negative. C: Whole mounts of YFP-H retina were quadruply stained for YFP, Opn, vAChT and the indicated marker. Cells that were Opn, YFP, and marker triple-positive were identified (green, cyan and red, respectively in top panels) and imaged (YFP only, middle panels). Stratification of YFP-positive dendrites was then determined with reference to that of starburst amacrines (vAChT-positive, red in bottom panels). Arrows point to the same cell displayed in each panel. Results are representative of 7–10 cells per type from 5 mice. Scale bar = 50 μm.

    Journal: PLoS ONE

    Article Title: Four alpha ganglion cell types in mouse retina: Function, structure, and molecular signatures

    doi: 10.1371/journal.pone.0180091

    Figure Lengend Snippet: Molecular distinctions among αRGCs. A, B : Retinal whole mounts were stained with antibodies to Opn (green), plus antibodies to one of 3 POU-domain transcription factors (Brn3a, Brn3b or Brn3c; red in A ) or one of 3 calcium binding proteins (parvalbumin [PV], calbindin or calretinin; red in B ). Brn3b and PV mark most αRGCs whereas Brn3a, Brn3c and calbindin mark subsets; most αRGCs are calretinin-negative. C: Whole mounts of YFP-H retina were quadruply stained for YFP, Opn, vAChT and the indicated marker. Cells that were Opn, YFP, and marker triple-positive were identified (green, cyan and red, respectively in top panels) and imaged (YFP only, middle panels). Stratification of YFP-positive dendrites was then determined with reference to that of starburst amacrines (vAChT-positive, red in bottom panels). Arrows point to the same cell displayed in each panel. Results are representative of 7–10 cells per type from 5 mice. Scale bar = 50 μm.

    Article Snippet: The primary antibodies used were: anti-GFP (rabbit, Life Technologies; chick, Abcam); mouse anti-Nonphosphoneurofilament H (SMI-32, Covance); goat anti-Osteopontin (R & D Systems); rabbit anti-Parvalbumin, rabbit anti-Calbindin, and mouse anti-Calretinin (all from Swant); anti-vAChT (goat, Promega; guinea pig, Millipore); goat anti-ChAT (Millipore); mouse anti-Brn3a (Millipore); goat anti-Brn3 (raised against Brn3b, Santa Cruz Biotechnology) and mouse anti-Brn3c (Santa Cruz Biotechnology).

    Techniques: Staining, Binding Assay, Marker, Mouse Assay

    Reconstitution of the mast cell pool in the lungs of Kit W-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. ( a ) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. ( b ) Representative images of toluidine blue staining of mast cells in the lungs of Kit W-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in Kit W-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm 2 ) in mid-lobe sections of each mouse lung and 4 mice for each condition. ( c ) Representative images of TuJ1 staining of airways (0.1–0.3 mm 2 in luminal area) from PBS- and OVA-exposed Kit W-sh/W-sh mice that received intra-tracheal instillation of WT or NT4 −/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed Kit W-sh/W-sh mice with and without adoptive transfer of WT and NT4 −/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. ( d ) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA-exposed wild type mice were assayed for the levels of VAChT. Each lane represents 1 mouse. GAPDH was loading control. Data were normalized to PBS control mice. N=12. ( e ) Western blot analysis for cholinergic innervation in lungs of Kit W-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed Kit W-sh/W-sh mice. ( f ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and Kit W-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and Kit W-sh/W-sh mice following OVA exposure were marked. ( g ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed Kit W-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Statistically significant differences between WT and NT4 −/− mast cell transfer were marked. The same results of OVA-exposed Kit W-sh/W-sh mice were plotted in both ( f ) and ( g ). *P

    Journal: Mucosal immunology

    Article Title: Mast cell-derived neurotrophin 4 mediates allergen-induced airway hyperinnervation in early life

    doi: 10.1038/mi.2016.11

    Figure Lengend Snippet: Reconstitution of the mast cell pool in the lungs of Kit W-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. ( a ) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. ( b ) Representative images of toluidine blue staining of mast cells in the lungs of Kit W-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in Kit W-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm 2 ) in mid-lobe sections of each mouse lung and 4 mice for each condition. ( c ) Representative images of TuJ1 staining of airways (0.1–0.3 mm 2 in luminal area) from PBS- and OVA-exposed Kit W-sh/W-sh mice that received intra-tracheal instillation of WT or NT4 −/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed Kit W-sh/W-sh mice with and without adoptive transfer of WT and NT4 −/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. ( d ) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA-exposed wild type mice were assayed for the levels of VAChT. Each lane represents 1 mouse. GAPDH was loading control. Data were normalized to PBS control mice. N=12. ( e ) Western blot analysis for cholinergic innervation in lungs of Kit W-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed Kit W-sh/W-sh mice. ( f ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and Kit W-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and Kit W-sh/W-sh mice following OVA exposure were marked. ( g ) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed Kit W-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Statistically significant differences between WT and NT4 −/− mast cell transfer were marked. The same results of OVA-exposed Kit W-sh/W-sh mice were plotted in both ( f ) and ( g ). *P

    Article Snippet: Primary antibodies for VAChT (1:100, Abcam #AB68986) and GAPDH (1:10,000, Abcam #AB8245), NT4 (1:100, ANT004, Alomone labs, Israel) were applied in blocking buffer.

    Techniques: Mouse Assay, Adoptive Transfer Assay, Staining, Western Blot

    NRG-1 injection regulated protein levels of neural remodeling markers in rat ischemic myocardia. Target protein expression was analyzed by western blot assay with antibodies against TH, GAP43, NGF, CHAT, and VACHT relative to GAPDH. MI: Myocardial infarction; N: neuregulin-1; TH: tyrosine hydroxylase; GAP43: growth associated protein-43; NGF: nerve growth factor; CHAT: choline acetyltransferase; VACHT: vesicular acetylcholine transporter; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

    Journal: Neural Regeneration Research

    Article Title: Effects of neuregulin-1 on autonomic nervous system remodeling post-myocardial infarction in a rat model

    doi: 10.4103/1673-5374.219054

    Figure Lengend Snippet: NRG-1 injection regulated protein levels of neural remodeling markers in rat ischemic myocardia. Target protein expression was analyzed by western blot assay with antibodies against TH, GAP43, NGF, CHAT, and VACHT relative to GAPDH. MI: Myocardial infarction; N: neuregulin-1; TH: tyrosine hydroxylase; GAP43: growth associated protein-43; NGF: nerve growth factor; CHAT: choline acetyltransferase; VACHT: vesicular acetylcholine transporter; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

    Article Snippet: The membranes were blocked by incubation with 5% bovine serum albumin in a TBS-Tween buffer (10 mM Tris-HCl, 150 mM NaCl, and 0.5% Tween-20) for 1 hour at room temperature, and subsequently, incubated with different primary antibodies: rabbit anti-TH (Santa, Shanghai, China), anti-GAP43 (1:1,000; Abcam, Cambridge, UK), anti-NGF (1:500; Abcam), anti-CHAT (1:500; Bioss, Beijing, China), anti-VACHT (1:500; Abcam), or anti-GAPDH (1:10,000; Abcam) overnight at 4°C.

    Techniques: Injection, Expressing, Western Blot