vglut1  (Alomone Labs)


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    Structured Review

    Alomone Labs vglut1
    Morphological analysis of the neuronal and glial components in Cdc42ep4 fl/fl and Cdc42ep4 −/− cerebellar cortices. ( a ) Double-label IF of WT and KO cerebellar cortices for a Purkinje cell marker Car8 (red) and a parallel fibre (that is, granule cell) marker <t>VGluT1</t> (top, green) or a climbing fibre marker VGluT2 (bottom, green). No obvious morphological anomaly, including aberrant CF–PC innervation, was found in the major neuronal components of KO-derived samples. Scale bar, 20 μm. ( b ) Transmission electron microscopy images of WT and KO molecular layers. No obvious ultrastructural difference was found between the genotypes. PF, parallel fibre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. Scale bar, 200 nm. ( c ) Cumulative histogram of PSD length of the PF–PC synapses, showing no significant difference between the genotypes ( n =92 synapses from two littermates for each genotype, NS, P > 0.05 by Kolmogorov–Smirnov test). ( d ) Quantitative immunoblot of WT and KO cerebellar PSD fractions for GluA1, GluA2 and GluA4 (the major subunits of the AMPARs), each normalized with PSD-95. There was no significant quantitative difference by genotype ( n =3, NS, P > 0.05 by t -test).
    Vglut1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance"

    Article Title: A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

    Journal: Nature Communications

    doi: 10.1038/ncomms10090

    Morphological analysis of the neuronal and glial components in Cdc42ep4 fl/fl and Cdc42ep4 −/− cerebellar cortices. ( a ) Double-label IF of WT and KO cerebellar cortices for a Purkinje cell marker Car8 (red) and a parallel fibre (that is, granule cell) marker VGluT1 (top, green) or a climbing fibre marker VGluT2 (bottom, green). No obvious morphological anomaly, including aberrant CF–PC innervation, was found in the major neuronal components of KO-derived samples. Scale bar, 20 μm. ( b ) Transmission electron microscopy images of WT and KO molecular layers. No obvious ultrastructural difference was found between the genotypes. PF, parallel fibre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. Scale bar, 200 nm. ( c ) Cumulative histogram of PSD length of the PF–PC synapses, showing no significant difference between the genotypes ( n =92 synapses from two littermates for each genotype, NS, P > 0.05 by Kolmogorov–Smirnov test). ( d ) Quantitative immunoblot of WT and KO cerebellar PSD fractions for GluA1, GluA2 and GluA4 (the major subunits of the AMPARs), each normalized with PSD-95. There was no significant quantitative difference by genotype ( n =3, NS, P > 0.05 by t -test).
    Figure Legend Snippet: Morphological analysis of the neuronal and glial components in Cdc42ep4 fl/fl and Cdc42ep4 −/− cerebellar cortices. ( a ) Double-label IF of WT and KO cerebellar cortices for a Purkinje cell marker Car8 (red) and a parallel fibre (that is, granule cell) marker VGluT1 (top, green) or a climbing fibre marker VGluT2 (bottom, green). No obvious morphological anomaly, including aberrant CF–PC innervation, was found in the major neuronal components of KO-derived samples. Scale bar, 20 μm. ( b ) Transmission electron microscopy images of WT and KO molecular layers. No obvious ultrastructural difference was found between the genotypes. PF, parallel fibre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. Scale bar, 200 nm. ( c ) Cumulative histogram of PSD length of the PF–PC synapses, showing no significant difference between the genotypes ( n =92 synapses from two littermates for each genotype, NS, P > 0.05 by Kolmogorov–Smirnov test). ( d ) Quantitative immunoblot of WT and KO cerebellar PSD fractions for GluA1, GluA2 and GluA4 (the major subunits of the AMPARs), each normalized with PSD-95. There was no significant quantitative difference by genotype ( n =3, NS, P > 0.05 by t -test).

    Techniques Used: Marker, Derivative Assay, Transmission Assay, Electron Microscopy

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    Alomone Labs kir4 1 ab
    In vivo intravitreal application of <t>Kir4.1-Ab:carrier</t> reduces STR and PhNR electronegative ERG potentials in RCS rat. ( A ) STR and PhNR were diminished in the eye of a 15-wk-old rat injected with Kir4.1-Ab:carrier (red) relative to the contralateral IgG:carrier control eye (black). ( B ) Light-adapted ERG responses (stimulus intensity 0.6 log cd-s/m 2 ) before (black) and 2 h after injection in a representative RCS rat at 14 wk of age. One eye was injected with Kir4.1-Ab:carrier (red) and the contralateral control eye with rabbit IgG:carrier (gray). PhNR amplitudes were decreased by 29 ± 5% ( n = 3) compared with the same eye baseline, whereas no change in PhNR amplitude was seen in the contralateral control eyes.
    Kir4 1 Ab, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs kir4 1
    Posthypoxic effects in retinal water/ion channels, gliosis and cell death. (a) AQP-4 immunostaining of retina in control (a), 1h (b) and 18h (c) animals. (d-f) <t>Kir4.1</t> immunostaining. (g-i) GFAP immunostaining (green) and DAPI nuclei labeling (blue). (j-l) TUNEL (red, arrows) and DAPI (blue). (m, n) Bar graph of quantification of AQP-4 (m) and Kir4.1 (n) expression levels in the retina and correlation with retinal thickness. (o) Bar graph of quantification of GFAP + processes across the IPL and correlation with retinal thickness. (p) Bar graph of quantification of the number of TUNEL + cells in each retina layer per retinal section. GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; OPL: outer plexiform layer, ONL: outer nuclear layer.
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    92
    Alomone Labs rabbit anti trpv5
    Channel characteristics of wild-type and mutant <t>TRPV5.</t> ( A ) Whole-cell currents in TRPV5-WT (V5-WT) and TRPV5-682P (V5-S682P) injected Xenopus oocytes recorded in response to 300 ms test pulses to various potentials (from −100 to +60 mV in 10 mV increments). Holding potential, 0 mV (N = 5). ( B ) Mean current-voltage relationships for TRPV5-WT and TRPV5-682P channels (N = 5). These current-voltage relationships are similar to those reported for TRPV5 channels [62] . ( C ) Mean whole-cell tail currents measured in TRPV5-WT and TRPV5-682P injected Xenopus oocytes during test potentials applied in 10 mV increments from −70 to +40 mV after a pre-pulse to −100 mV in TRPV5-WT and TRPV5-682P channels (N = 5). ( D ) Time-dependent inhibition of TRPV-WT and TRPV5-682P whole-cell currents. Oocytes were stimulated every 1 s. The peak current amplitude was normalised to that recorded during the first pulse (N = 4). ( E ) Representative trace of Fura-2 ratio in HEK293 cells transiently transfected with an empty EGFP vector (mock), or EGFP-tagged TRPV5-WT or TRPV5-S682P. Cells expressing EGFP were selected and monitored for changes in intracellular Ca 2+ levels when extracellular Ca 2+ concentrations were varied from 1.4 mM Ca 2+ to 0 mM Ca 2+ (2 mM EDTA) and 1.4 mM Ca 2+ which was facilitated by superfusion. ( F ) Fura-2 levels under resting conditions (t0), minimal Fura-2 ratio after EDTA treatment (tmin) and peak level (tmax) upon administration of 1.4 mM Ca 2+ after EDTA treatment. Average data of cells transfected with the empty vector (n = 7), TRPV5-wt (n = 24) and TRPV5-S682P (N = 24) from at least three independent experiments. * p
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    Image Search Results


    In vivo intravitreal application of Kir4.1-Ab:carrier reduces STR and PhNR electronegative ERG potentials in RCS rat. ( A ) STR and PhNR were diminished in the eye of a 15-wk-old rat injected with Kir4.1-Ab:carrier (red) relative to the contralateral IgG:carrier control eye (black). ( B ) Light-adapted ERG responses (stimulus intensity 0.6 log cd-s/m 2 ) before (black) and 2 h after injection in a representative RCS rat at 14 wk of age. One eye was injected with Kir4.1-Ab:carrier (red) and the contralateral control eye with rabbit IgG:carrier (gray). PhNR amplitudes were decreased by 29 ± 5% ( n = 3) compared with the same eye baseline, whereas no change in PhNR amplitude was seen in the contralateral control eyes.

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

    Article Title: Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration

    doi: 10.1073/pnas.0913472107

    Figure Lengend Snippet: In vivo intravitreal application of Kir4.1-Ab:carrier reduces STR and PhNR electronegative ERG potentials in RCS rat. ( A ) STR and PhNR were diminished in the eye of a 15-wk-old rat injected with Kir4.1-Ab:carrier (red) relative to the contralateral IgG:carrier control eye (black). ( B ) Light-adapted ERG responses (stimulus intensity 0.6 log cd-s/m 2 ) before (black) and 2 h after injection in a representative RCS rat at 14 wk of age. One eye was injected with Kir4.1-Ab:carrier (red) and the contralateral control eye with rabbit IgG:carrier (gray). PhNR amplitudes were decreased by 29 ± 5% ( n = 3) compared with the same eye baseline, whereas no change in PhNR amplitude was seen in the contralateral control eyes.

    Article Snippet: Primary antibodies were polyclonal rabbit anti-human Kir2.1-Ab and Kir4.1-Ab (1:50 dilution; Alomone Labs), monoclonal mouse anti-human protein kinase C alpha (PKCα-Ab 1:200; Santa Cruz Biotechnology) to label rod BCs , monoclonal mouse anti-human protein kinase C beta (PKCβ-Ab, 1:200; Santa Cruz Biotechnology) to label cone BCs , polyclonal guinea-pig anti-rat vesicular glutamate transporter 1 (VGLUT1-Ab, 1:5,000; Chemicon) to label rod and cone BCs , polyclonal goat anti-human synapsin Ia/b-Ab (1:50; Santa Cruz Biotechnology) and IIa-Ab (1:100; Santa Cruz Biotechnology) as amacrine cell markers that label phosphoproteins of conventional synapses but not ribbon-containing terminals , and polyclonal GFAP-Ab (Sigma, 1:200) as Müller cell marker.

    Techniques: In Vivo, Injection

    IHC of rat retina shows Kir4.1 colocalization with Müller cell GFAP marker. ( A ) Conventional postmortem double labeling with Kir4.1-Ab (red) and GFAP-Ab (green) shows colocalization with Müller cells in a dystrophic 14 wk-old RCS retina. ( B ) In vivo intravitreal application of Kir4.1-Ab:carrier complex gives prominent labeling of Müller cell endfeet with extensions along Müller cell processes in the IPL (red) in a 15-wk-old dystrophic RCS rat and colocalizes with GFAP (green). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.

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

    Article Title: Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration

    doi: 10.1073/pnas.0913472107

    Figure Lengend Snippet: IHC of rat retina shows Kir4.1 colocalization with Müller cell GFAP marker. ( A ) Conventional postmortem double labeling with Kir4.1-Ab (red) and GFAP-Ab (green) shows colocalization with Müller cells in a dystrophic 14 wk-old RCS retina. ( B ) In vivo intravitreal application of Kir4.1-Ab:carrier complex gives prominent labeling of Müller cell endfeet with extensions along Müller cell processes in the IPL (red) in a 15-wk-old dystrophic RCS rat and colocalizes with GFAP (green). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.

    Article Snippet: Primary antibodies were polyclonal rabbit anti-human Kir2.1-Ab and Kir4.1-Ab (1:50 dilution; Alomone Labs), monoclonal mouse anti-human protein kinase C alpha (PKCα-Ab 1:200; Santa Cruz Biotechnology) to label rod BCs , monoclonal mouse anti-human protein kinase C beta (PKCβ-Ab, 1:200; Santa Cruz Biotechnology) to label cone BCs , polyclonal guinea-pig anti-rat vesicular glutamate transporter 1 (VGLUT1-Ab, 1:5,000; Chemicon) to label rod and cone BCs , polyclonal goat anti-human synapsin Ia/b-Ab (1:50; Santa Cruz Biotechnology) and IIa-Ab (1:100; Santa Cruz Biotechnology) as amacrine cell markers that label phosphoproteins of conventional synapses but not ribbon-containing terminals , and polyclonal GFAP-Ab (Sigma, 1:200) as Müller cell marker.

    Techniques: Immunohistochemistry, Marker, Labeling, In Vivo

    Immunohistochemical analysis of Kir4.1- and GFAP-immunoreactivity (IR)-positive cells in pilocarpine-inducedTLE rats. (A) Schematic illustrations of the brain sections selected for quantitative analysis of Kir4.1- and GFAP-IR-positive cells. Squares in each section indicate the area analyzed for counting of Kir4.1- and GFAP-IR-positive cells. The distance from the Bregma is shown on the bottom of each section. MC, motor cortex; SC, sensory cortex; AID, agranular insular cortex; Pir, piriform cortex; dmST, vmST, dlST and vlST, dorsomedial, ventromedial, dorsolateral, and ventrolateral striatum, respectively; AcbC and AcbSh, core and shell regions of nucleus accumbens, respectively; Ect-PRh, ectorhinal–perirhinal cortex; MePV and MePD, medial amygdaloid nucleus, posteroventral and posterodorsal part; BLP, basolateral amygdaloid nucleus, posterior part; BMP, basomedial amygdaloid nucleus, posterior part; PMCo, posteromedial cortical amygdaloid nucleus; CA1, CA3, and DG: CA1, CA3, and dentate gyrus of the hippocampus. (B) Representative photographs illustrating the Kir4.1 (upper panels)- and GFAP (lower panels)-positive cells in the sensory cortex (SC) and the medial amygdaloid nucleus, posterodorsal part (MePD). Scale bar: 50 μ m.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expressional analysis of the astrocytic Kir4.1 channel in a pilocarpine-induced temporal lobe epilepsy model

    doi: 10.3389/fncel.2013.00104

    Figure Lengend Snippet: Immunohistochemical analysis of Kir4.1- and GFAP-immunoreactivity (IR)-positive cells in pilocarpine-inducedTLE rats. (A) Schematic illustrations of the brain sections selected for quantitative analysis of Kir4.1- and GFAP-IR-positive cells. Squares in each section indicate the area analyzed for counting of Kir4.1- and GFAP-IR-positive cells. The distance from the Bregma is shown on the bottom of each section. MC, motor cortex; SC, sensory cortex; AID, agranular insular cortex; Pir, piriform cortex; dmST, vmST, dlST and vlST, dorsomedial, ventromedial, dorsolateral, and ventrolateral striatum, respectively; AcbC and AcbSh, core and shell regions of nucleus accumbens, respectively; Ect-PRh, ectorhinal–perirhinal cortex; MePV and MePD, medial amygdaloid nucleus, posteroventral and posterodorsal part; BLP, basolateral amygdaloid nucleus, posterior part; BMP, basomedial amygdaloid nucleus, posterior part; PMCo, posteromedial cortical amygdaloid nucleus; CA1, CA3, and DG: CA1, CA3, and dentate gyrus of the hippocampus. (B) Representative photographs illustrating the Kir4.1 (upper panels)- and GFAP (lower panels)-positive cells in the sensory cortex (SC) and the medial amygdaloid nucleus, posterodorsal part (MePD). Scale bar: 50 μ m.

    Article Snippet: The primary antibodies used were a rabbit polyclonal antibody against Kir4.1 (1:500, Alomone Labs., Jerusalem, Israel), a goat polyclonal antibody against Kir5.1 (N-12; 1:400, Santa Cruz Biotechnology), a goat polyclonal antibody against Kir2.1 (1:400, Santa Cruz Biotechnology) and mouse monoclonal antibodies against β-actin (1:1000, Sigma-Aldrich).

    Techniques: Immunohistochemistry

    Western blot analysis for Kir4.1, Kir5.1 and Kir2.1 expression in pilocarpine-inducedTLE rats. (A) Representative Western blots visualizing Kir4.1, Kir5.1, and Kir2.1 expression in the frontal cortex (fCx), occipito-temporal cortex (otCx), striatum (St), and hypothalamus (Ht). (B–D) Regional expression of Kir4.1 (B) , Kir5.1 (C) , and Kir2.1 (D) in pilocarpine-induced TLE rats. Kir expression was expressed as relative optical density (ROD) to β-actin. fCx, frontal cortex; ptCx, parieto-temporal cortex; otCx, occipito-temporal cortex; St, striatum; Hpc, hippocampus; Th, thalamus; Ht, hypothalamus; Mid, midbrain; P/MO, pons/medulla oblongata; Cer, cerebellum. Each column represents the mean ± SEM of four animals. * P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expressional analysis of the astrocytic Kir4.1 channel in a pilocarpine-induced temporal lobe epilepsy model

    doi: 10.3389/fncel.2013.00104

    Figure Lengend Snippet: Western blot analysis for Kir4.1, Kir5.1 and Kir2.1 expression in pilocarpine-inducedTLE rats. (A) Representative Western blots visualizing Kir4.1, Kir5.1, and Kir2.1 expression in the frontal cortex (fCx), occipito-temporal cortex (otCx), striatum (St), and hypothalamus (Ht). (B–D) Regional expression of Kir4.1 (B) , Kir5.1 (C) , and Kir2.1 (D) in pilocarpine-induced TLE rats. Kir expression was expressed as relative optical density (ROD) to β-actin. fCx, frontal cortex; ptCx, parieto-temporal cortex; otCx, occipito-temporal cortex; St, striatum; Hpc, hippocampus; Th, thalamus; Ht, hypothalamus; Mid, midbrain; P/MO, pons/medulla oblongata; Cer, cerebellum. Each column represents the mean ± SEM of four animals. * P

    Article Snippet: The primary antibodies used were a rabbit polyclonal antibody against Kir4.1 (1:500, Alomone Labs., Jerusalem, Israel), a goat polyclonal antibody against Kir5.1 (N-12; 1:400, Santa Cruz Biotechnology), a goat polyclonal antibody against Kir2.1 (1:400, Santa Cruz Biotechnology) and mouse monoclonal antibodies against β-actin (1:1000, Sigma-Aldrich).

    Techniques: Western Blot, Expressing

    Topographical expression of Kir4.1 and GFAP in the cortical regions of pilocarpine-inducedTLE rats. (A,B) Number of Kir4.1 (A) - or GFAP (B) -immunoreactivity (IR)-positive cells. (C) Relative Kir4.1 expression ratios in astrocytes. A pair of successive slices in each region from the same animal was stained with anti-Kir4.1 or anti-GFAP antibody. The Kir4.1 expression ratios were calculated as the ratios of Kir4.1-positive astrocytes relative to the total number of astrocytes (Kir4.1-positive cells/GFAP-positive cells) in each animal. MC, motor cortex; SC, sensory cortex; AID, agranular insular cortex, dorsal part; Ect-PRh, ectorhinal-perirhinal cortex; Pir, piriform cortex. Each column represents the mean ± S.E.M. of seven animals. * P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expressional analysis of the astrocytic Kir4.1 channel in a pilocarpine-induced temporal lobe epilepsy model

    doi: 10.3389/fncel.2013.00104

    Figure Lengend Snippet: Topographical expression of Kir4.1 and GFAP in the cortical regions of pilocarpine-inducedTLE rats. (A,B) Number of Kir4.1 (A) - or GFAP (B) -immunoreactivity (IR)-positive cells. (C) Relative Kir4.1 expression ratios in astrocytes. A pair of successive slices in each region from the same animal was stained with anti-Kir4.1 or anti-GFAP antibody. The Kir4.1 expression ratios were calculated as the ratios of Kir4.1-positive astrocytes relative to the total number of astrocytes (Kir4.1-positive cells/GFAP-positive cells) in each animal. MC, motor cortex; SC, sensory cortex; AID, agranular insular cortex, dorsal part; Ect-PRh, ectorhinal-perirhinal cortex; Pir, piriform cortex. Each column represents the mean ± S.E.M. of seven animals. * P

    Article Snippet: The primary antibodies used were a rabbit polyclonal antibody against Kir4.1 (1:500, Alomone Labs., Jerusalem, Israel), a goat polyclonal antibody against Kir5.1 (N-12; 1:400, Santa Cruz Biotechnology), a goat polyclonal antibody against Kir2.1 (1:400, Santa Cruz Biotechnology) and mouse monoclonal antibodies against β-actin (1:1000, Sigma-Aldrich).

    Techniques: Expressing, Staining

    Topographical expression of Kir4.1 and GFAP in the basal ganglia and limbic regions of pilocarpine-inducedTLE rats. (A,B) Number of Kir4.1 (A) - or GFAP (B) -immunoreactivity (IR)-positive cells. (C) Relative Kir4.1 expression ratios in astrocytes. A pair of successive slices in each region from the same animal was stained with anti-Kir4.1 or anti-GFAP antibody. The Kir4.1 expression ratios were calculated as the ratios of Kir4.1-positive astrocytes relative to the total number of astrocytes (Kir4.1Kir4.1-positive cells/GFAP-positive cells) in each animal. dmST, vmST, dlST, and vlST, dorsomedial, ventromedial, dorsolateral, and ventrolateral striatum, respectively; AcbC and AcbSh, core and shell regions of the nucleus accumbens, respectively; MePV and MePD, medial amygdaloid nucleus, posteroventral and posterodorsal part; BLP, basolateral amygdaloid nucleus, posterior part; BMP, basomedial amygdaloid nucleus, posterior part; PMCo, posteromedial cortical amygdaloid nucleus; CA1, CA3, and DG, CA1, CA3, and dentate gyrus of the hippocampus. Each column represents the mean ± SEM of seven animals. * P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expressional analysis of the astrocytic Kir4.1 channel in a pilocarpine-induced temporal lobe epilepsy model

    doi: 10.3389/fncel.2013.00104

    Figure Lengend Snippet: Topographical expression of Kir4.1 and GFAP in the basal ganglia and limbic regions of pilocarpine-inducedTLE rats. (A,B) Number of Kir4.1 (A) - or GFAP (B) -immunoreactivity (IR)-positive cells. (C) Relative Kir4.1 expression ratios in astrocytes. A pair of successive slices in each region from the same animal was stained with anti-Kir4.1 or anti-GFAP antibody. The Kir4.1 expression ratios were calculated as the ratios of Kir4.1-positive astrocytes relative to the total number of astrocytes (Kir4.1Kir4.1-positive cells/GFAP-positive cells) in each animal. dmST, vmST, dlST, and vlST, dorsomedial, ventromedial, dorsolateral, and ventrolateral striatum, respectively; AcbC and AcbSh, core and shell regions of the nucleus accumbens, respectively; MePV and MePD, medial amygdaloid nucleus, posteroventral and posterodorsal part; BLP, basolateral amygdaloid nucleus, posterior part; BMP, basomedial amygdaloid nucleus, posterior part; PMCo, posteromedial cortical amygdaloid nucleus; CA1, CA3, and DG, CA1, CA3, and dentate gyrus of the hippocampus. Each column represents the mean ± SEM of seven animals. * P

    Article Snippet: The primary antibodies used were a rabbit polyclonal antibody against Kir4.1 (1:500, Alomone Labs., Jerusalem, Israel), a goat polyclonal antibody against Kir5.1 (N-12; 1:400, Santa Cruz Biotechnology), a goat polyclonal antibody against Kir2.1 (1:400, Santa Cruz Biotechnology) and mouse monoclonal antibodies against β-actin (1:1000, Sigma-Aldrich).

    Techniques: Expressing, Staining

    Posthypoxic effects in retinal water/ion channels, gliosis and cell death. (a) AQP-4 immunostaining of retina in control (a), 1h (b) and 18h (c) animals. (d-f) Kir4.1 immunostaining. (g-i) GFAP immunostaining (green) and DAPI nuclei labeling (blue). (j-l) TUNEL (red, arrows) and DAPI (blue). (m, n) Bar graph of quantification of AQP-4 (m) and Kir4.1 (n) expression levels in the retina and correlation with retinal thickness. (o) Bar graph of quantification of GFAP + processes across the IPL and correlation with retinal thickness. (p) Bar graph of quantification of the number of TUNEL + cells in each retina layer per retinal section. GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; OPL: outer plexiform layer, ONL: outer nuclear layer.

    Journal: PLoS ONE

    Article Title: Hypoxia-induced inflammation: Profiling the first 24-hour posthypoxic plasma and central nervous system changes

    doi: 10.1371/journal.pone.0246681

    Figure Lengend Snippet: Posthypoxic effects in retinal water/ion channels, gliosis and cell death. (a) AQP-4 immunostaining of retina in control (a), 1h (b) and 18h (c) animals. (d-f) Kir4.1 immunostaining. (g-i) GFAP immunostaining (green) and DAPI nuclei labeling (blue). (j-l) TUNEL (red, arrows) and DAPI (blue). (m, n) Bar graph of quantification of AQP-4 (m) and Kir4.1 (n) expression levels in the retina and correlation with retinal thickness. (o) Bar graph of quantification of GFAP + processes across the IPL and correlation with retinal thickness. (p) Bar graph of quantification of the number of TUNEL + cells in each retina layer per retinal section. GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; OPL: outer plexiform layer, ONL: outer nuclear layer.

    Article Snippet: Retina immunostainingRetinae were immunostained with primary antibodies to detect and AQP-4 (1:50, mouse; catalog number sc-32739, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA), Kir4.1 (1:200, rabbit, catalog number APC-035, Alomone Labs, Jerusalem, Israel) and glial fibrillary acidic protein (GFAP) (1:1000, rabbit; catalog number ab7260; Abcam, Cambridge, MA, USA).

    Techniques: Immunostaining, Labeling, TUNEL Assay, Expressing

    Channel characteristics of wild-type and mutant TRPV5. ( A ) Whole-cell currents in TRPV5-WT (V5-WT) and TRPV5-682P (V5-S682P) injected Xenopus oocytes recorded in response to 300 ms test pulses to various potentials (from −100 to +60 mV in 10 mV increments). Holding potential, 0 mV (N = 5). ( B ) Mean current-voltage relationships for TRPV5-WT and TRPV5-682P channels (N = 5). These current-voltage relationships are similar to those reported for TRPV5 channels [62] . ( C ) Mean whole-cell tail currents measured in TRPV5-WT and TRPV5-682P injected Xenopus oocytes during test potentials applied in 10 mV increments from −70 to +40 mV after a pre-pulse to −100 mV in TRPV5-WT and TRPV5-682P channels (N = 5). ( D ) Time-dependent inhibition of TRPV-WT and TRPV5-682P whole-cell currents. Oocytes were stimulated every 1 s. The peak current amplitude was normalised to that recorded during the first pulse (N = 4). ( E ) Representative trace of Fura-2 ratio in HEK293 cells transiently transfected with an empty EGFP vector (mock), or EGFP-tagged TRPV5-WT or TRPV5-S682P. Cells expressing EGFP were selected and monitored for changes in intracellular Ca 2+ levels when extracellular Ca 2+ concentrations were varied from 1.4 mM Ca 2+ to 0 mM Ca 2+ (2 mM EDTA) and 1.4 mM Ca 2+ which was facilitated by superfusion. ( F ) Fura-2 levels under resting conditions (t0), minimal Fura-2 ratio after EDTA treatment (tmin) and peak level (tmax) upon administration of 1.4 mM Ca 2+ after EDTA treatment. Average data of cells transfected with the empty vector (n = 7), TRPV5-wt (n = 24) and TRPV5-S682P (N = 24) from at least three independent experiments. * p

    Journal: PLoS ONE

    Article Title: Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel, TRPV5

    doi: 10.1371/journal.pone.0055412

    Figure Lengend Snippet: Channel characteristics of wild-type and mutant TRPV5. ( A ) Whole-cell currents in TRPV5-WT (V5-WT) and TRPV5-682P (V5-S682P) injected Xenopus oocytes recorded in response to 300 ms test pulses to various potentials (from −100 to +60 mV in 10 mV increments). Holding potential, 0 mV (N = 5). ( B ) Mean current-voltage relationships for TRPV5-WT and TRPV5-682P channels (N = 5). These current-voltage relationships are similar to those reported for TRPV5 channels [62] . ( C ) Mean whole-cell tail currents measured in TRPV5-WT and TRPV5-682P injected Xenopus oocytes during test potentials applied in 10 mV increments from −70 to +40 mV after a pre-pulse to −100 mV in TRPV5-WT and TRPV5-682P channels (N = 5). ( D ) Time-dependent inhibition of TRPV-WT and TRPV5-682P whole-cell currents. Oocytes were stimulated every 1 s. The peak current amplitude was normalised to that recorded during the first pulse (N = 4). ( E ) Representative trace of Fura-2 ratio in HEK293 cells transiently transfected with an empty EGFP vector (mock), or EGFP-tagged TRPV5-WT or TRPV5-S682P. Cells expressing EGFP were selected and monitored for changes in intracellular Ca 2+ levels when extracellular Ca 2+ concentrations were varied from 1.4 mM Ca 2+ to 0 mM Ca 2+ (2 mM EDTA) and 1.4 mM Ca 2+ which was facilitated by superfusion. ( F ) Fura-2 levels under resting conditions (t0), minimal Fura-2 ratio after EDTA treatment (tmin) and peak level (tmax) upon administration of 1.4 mM Ca 2+ after EDTA treatment. Average data of cells transfected with the empty vector (n = 7), TRPV5-wt (n = 24) and TRPV5-S682P (N = 24) from at least three independent experiments. * p

    Article Snippet: For TRPV5 immuno-detection, 8- µm kidney cryosections were processed for immunofluorescence labelling as previously described ._ENREF_41 Kidney cryosections were co-stained with rabbit anti-TRPV5 (ACC-035, Alomone Labs, Jerusalem, Israel) and goat anti-AQP2 (sc-9882, Santa Cruz, Insight Biotechnology, Wembley, UK) polyclonal antibodies, or with goat anti-TRPV5 (sc-23379, Santa Cruz) and rabbit anti-NCC polyclonal antibodies, followed by the appropriate Alexa Fluor 488- or 594-conjugated secondary antibodies (Molecular Probes).

    Techniques: Mutagenesis, Injection, Mass Spectrometry, Inhibition, Transfection, Plasmid Preparation, Expressing

    Assessment of Renal Expression of Calcium Regulatory Genes and Proteins. Renal expression of ( A ) Trpv5 , ( B ) Trpv6 and ( C ) Cyp24a1 in wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice (n = 6/group) were assessed by quantitative real-time PCR. All data were normalised to levels of the housekeeping gene Gapdh and wild-type values are expressed as 1. Histogram data are presented as mean ± SEM. # p

    Journal: PLoS ONE

    Article Title: Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel, TRPV5

    doi: 10.1371/journal.pone.0055412

    Figure Lengend Snippet: Assessment of Renal Expression of Calcium Regulatory Genes and Proteins. Renal expression of ( A ) Trpv5 , ( B ) Trpv6 and ( C ) Cyp24a1 in wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice (n = 6/group) were assessed by quantitative real-time PCR. All data were normalised to levels of the housekeeping gene Gapdh and wild-type values are expressed as 1. Histogram data are presented as mean ± SEM. # p

    Article Snippet: For TRPV5 immuno-detection, 8- µm kidney cryosections were processed for immunofluorescence labelling as previously described ._ENREF_41 Kidney cryosections were co-stained with rabbit anti-TRPV5 (ACC-035, Alomone Labs, Jerusalem, Israel) and goat anti-AQP2 (sc-9882, Santa Cruz, Insight Biotechnology, Wembley, UK) polyclonal antibodies, or with goat anti-TRPV5 (sc-23379, Santa Cruz) and rabbit anti-NCC polyclonal antibodies, followed by the appropriate Alexa Fluor 488- or 594-conjugated secondary antibodies (Molecular Probes).

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

    Histological and immunohistochemical assessment of kidneys from HCALC1 mice. Representative images in Trpv5 +/+ (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice of: ( A ) Masson's trichrome staining of renal cortex showing areas of interstitial fibrosis in Trpv5 682P/+ and Trpv5 682P/682P mice (light blue), ( B ) anti-CD3-labelling (green) showing a large number of T-lymphocytes present in the interstitial regions of the Trpv5 682P/+ and Trpv5 682P/682P mouse kidneys, ( C ) TUNEL-labelling (green) of the renal cortex showing the presence of tubular cell apoptosis in the Trpv5 682P/+ and Trpv5 682P/682P mouse kidneys. Scale bar = 50 µm. ( D ) Immunohistochemical images of kidney sections from wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice, co-stained for TRPV5 (green) and NCC (red). * denotes co-localisation. Scale bar = 50 µm. ( E ) Kidney sections from wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice, co-stained for TRPV5 (green) and AQP2 (red). * denotes co-localisation. Scale bar = 50 µm.

    Journal: PLoS ONE

    Article Title: Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel, TRPV5

    doi: 10.1371/journal.pone.0055412

    Figure Lengend Snippet: Histological and immunohistochemical assessment of kidneys from HCALC1 mice. Representative images in Trpv5 +/+ (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice of: ( A ) Masson's trichrome staining of renal cortex showing areas of interstitial fibrosis in Trpv5 682P/+ and Trpv5 682P/682P mice (light blue), ( B ) anti-CD3-labelling (green) showing a large number of T-lymphocytes present in the interstitial regions of the Trpv5 682P/+ and Trpv5 682P/682P mouse kidneys, ( C ) TUNEL-labelling (green) of the renal cortex showing the presence of tubular cell apoptosis in the Trpv5 682P/+ and Trpv5 682P/682P mouse kidneys. Scale bar = 50 µm. ( D ) Immunohistochemical images of kidney sections from wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice, co-stained for TRPV5 (green) and NCC (red). * denotes co-localisation. Scale bar = 50 µm. ( E ) Kidney sections from wild-type (wt), Trpv5 682P/+ (het) and Trpv5 682P/682P (hom) mice, co-stained for TRPV5 (green) and AQP2 (red). * denotes co-localisation. Scale bar = 50 µm.

    Article Snippet: For TRPV5 immuno-detection, 8- µm kidney cryosections were processed for immunofluorescence labelling as previously described ._ENREF_41 Kidney cryosections were co-stained with rabbit anti-TRPV5 (ACC-035, Alomone Labs, Jerusalem, Israel) and goat anti-AQP2 (sc-9882, Santa Cruz, Insight Biotechnology, Wembley, UK) polyclonal antibodies, or with goat anti-TRPV5 (sc-23379, Santa Cruz) and rabbit anti-NCC polyclonal antibodies, followed by the appropriate Alexa Fluor 488- or 594-conjugated secondary antibodies (Molecular Probes).

    Techniques: Immunohistochemistry, Mouse Assay, Staining, TUNEL Assay

    Hypercalciuria in HCALC1 ENU mutant mice and identification of a Trpv5 mutation. ( A ) Urine calcium/creatinine ratios in 23 G2 offspring of the HCALC1 founder male revealed that 10 of the 23 mice were hypercalciuric, consistent with an autosomal dominant inheritance. Bar, mean calcium/creatinine values. ( B ) Haplotype analysis of 89 G2 mice (39 hypercalciuric and 50 normocalciuric) was initially undertaken separately in the hypercalciuric and normocalciuric mice, as the penetrance of HCALC1 was unknown. Haplotype analysis of the hypercalciuric mice localised Hcalc1 to a 17.38 Mb interval on chromosome 6, flanked by rs13478688 and rs30110406 (broken double-headed arrow). Haplotype analysis using combined data for the hypercalciuric and normocalciuric mice identified the smaller interval, 11.94-Mb, flanked by rs13478709 and rs30110406 (solid double-headed arrow). The Hcalc1 locus is inherited with the C57BL/6J haplotype from the F1 founder male. Filled box, C57BL/6J allele; and open box, C3H/HeH allele. Number of mice observed for each haplotype is shown beneath each column. ( C ) DNA sequence analysis of Trpv5 identified a heterozygous T to C transition in codon 682 in hypercalciuric mice predicted to alter a wild-type serine (Ser) to a mutant proline (Pro). This mutation resulted in gain of a Bsa JI restriction enzyme site that was used to confirm the presence of the mutation in the 39 hypercalciuric mice (n = 3 shown) and its absence in the 50 normocalciuric mice (n = 3 shown). wt, wild-type; m, mutant. ( D ) Amino acid sequence alignment revealed evolutionary conservation of the wild-type mouse TRPV5 serine (S) residue at codon 682 (arrowed) in 5 species, as well as in mouse TRPV6 (mTrpv6). Identical residues are shaded black and conservative changes are shaded grey.

    Journal: PLoS ONE

    Article Title: Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel, TRPV5

    doi: 10.1371/journal.pone.0055412

    Figure Lengend Snippet: Hypercalciuria in HCALC1 ENU mutant mice and identification of a Trpv5 mutation. ( A ) Urine calcium/creatinine ratios in 23 G2 offspring of the HCALC1 founder male revealed that 10 of the 23 mice were hypercalciuric, consistent with an autosomal dominant inheritance. Bar, mean calcium/creatinine values. ( B ) Haplotype analysis of 89 G2 mice (39 hypercalciuric and 50 normocalciuric) was initially undertaken separately in the hypercalciuric and normocalciuric mice, as the penetrance of HCALC1 was unknown. Haplotype analysis of the hypercalciuric mice localised Hcalc1 to a 17.38 Mb interval on chromosome 6, flanked by rs13478688 and rs30110406 (broken double-headed arrow). Haplotype analysis using combined data for the hypercalciuric and normocalciuric mice identified the smaller interval, 11.94-Mb, flanked by rs13478709 and rs30110406 (solid double-headed arrow). The Hcalc1 locus is inherited with the C57BL/6J haplotype from the F1 founder male. Filled box, C57BL/6J allele; and open box, C3H/HeH allele. Number of mice observed for each haplotype is shown beneath each column. ( C ) DNA sequence analysis of Trpv5 identified a heterozygous T to C transition in codon 682 in hypercalciuric mice predicted to alter a wild-type serine (Ser) to a mutant proline (Pro). This mutation resulted in gain of a Bsa JI restriction enzyme site that was used to confirm the presence of the mutation in the 39 hypercalciuric mice (n = 3 shown) and its absence in the 50 normocalciuric mice (n = 3 shown). wt, wild-type; m, mutant. ( D ) Amino acid sequence alignment revealed evolutionary conservation of the wild-type mouse TRPV5 serine (S) residue at codon 682 (arrowed) in 5 species, as well as in mouse TRPV6 (mTrpv6). Identical residues are shaded black and conservative changes are shaded grey.

    Article Snippet: For TRPV5 immuno-detection, 8- µm kidney cryosections were processed for immunofluorescence labelling as previously described ._ENREF_41 Kidney cryosections were co-stained with rabbit anti-TRPV5 (ACC-035, Alomone Labs, Jerusalem, Israel) and goat anti-AQP2 (sc-9882, Santa Cruz, Insight Biotechnology, Wembley, UK) polyclonal antibodies, or with goat anti-TRPV5 (sc-23379, Santa Cruz) and rabbit anti-NCC polyclonal antibodies, followed by the appropriate Alexa Fluor 488- or 594-conjugated secondary antibodies (Molecular Probes).

    Techniques: Mutagenesis, Mouse Assay, Sequencing