rabbit anti hcn4  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti hcn4
    LRMP and IRAG do not prevent cAMP binding to the CNBD A1-3: Representative current traces of <t>HCN4</t> in the absence or presence of LRMP or IRAG and the absence (black) or presence of 1 mM cAMP (red). Currents were elicited with 3 s hyperpolarizations to −150 mV followed by 3 s pulses to −50 mV. B: Average time to half maximal current at −150 mV of HCN4 in control (black) or presence of LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Error bars in this and C are SEM. Each individual observation is plotted as a circle and averages are plotted as squares. Number of observations for each dataset are given in parentheses. C: Average deactivation time constant of HCN4 at −50 mV in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as B . 4 outliers are not shown in C . * indicates P
    Rabbit Anti Hcn4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins"

    Article Title: Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins

    Journal: bioRxiv

    doi: 10.1101/2020.04.10.022483

    LRMP and IRAG do not prevent cAMP binding to the CNBD A1-3: Representative current traces of HCN4 in the absence or presence of LRMP or IRAG and the absence (black) or presence of 1 mM cAMP (red). Currents were elicited with 3 s hyperpolarizations to −150 mV followed by 3 s pulses to −50 mV. B: Average time to half maximal current at −150 mV of HCN4 in control (black) or presence of LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Error bars in this and C are SEM. Each individual observation is plotted as a circle and averages are plotted as squares. Number of observations for each dataset are given in parentheses. C: Average deactivation time constant of HCN4 at −50 mV in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as B . 4 outliers are not shown in C . * indicates P
    Figure Legend Snippet: LRMP and IRAG do not prevent cAMP binding to the CNBD A1-3: Representative current traces of HCN4 in the absence or presence of LRMP or IRAG and the absence (black) or presence of 1 mM cAMP (red). Currents were elicited with 3 s hyperpolarizations to −150 mV followed by 3 s pulses to −50 mV. B: Average time to half maximal current at −150 mV of HCN4 in control (black) or presence of LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Error bars in this and C are SEM. Each individual observation is plotted as a circle and averages are plotted as squares. Number of observations for each dataset are given in parentheses. C: Average deactivation time constant of HCN4 at −50 mV in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as B . 4 outliers are not shown in C . * indicates P

    Techniques Used: Binding Assay

    LRMP and IRAG have opposing effects on HCN4 function A1-6 : Representative whole cell HCN4 currents from HEK cells in the absence or presence of LRMP or IRAG with or without 1 mM cAMP in the patch pipette. Currents were elicited with 3 s hyperpolarizations to membrane potentials between −50 mV and −150 mV in 10 mV increments followed by 3 s pulses to −50 mV. Red traces are the currents at −110 mV. B, C: Average conductance-voltage relations for HCN4 in control conditions (black), the presence of LRMP (red), or the presence of IRAG (blue). GVs in the presence of 1 mM cAMP are shown by open symbols. Error bars in this and subsequent panels are SEM, N = 14-22 (See panel D ). Control HCN4 data in panel C are the same as those in panel B. D : Average V 1/2 values for HCN4 in HEK cells in the absence or presence of LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Each individual observation is plotted as a circle. Number of observations for each dataset is given in parentheses. Averages (± SEM) are plotted as squares. E : Average current density in response to a 3 s step to −150 mV of HCN4 in HEK cells in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as D . * indicates P
    Figure Legend Snippet: LRMP and IRAG have opposing effects on HCN4 function A1-6 : Representative whole cell HCN4 currents from HEK cells in the absence or presence of LRMP or IRAG with or without 1 mM cAMP in the patch pipette. Currents were elicited with 3 s hyperpolarizations to membrane potentials between −50 mV and −150 mV in 10 mV increments followed by 3 s pulses to −50 mV. Red traces are the currents at −110 mV. B, C: Average conductance-voltage relations for HCN4 in control conditions (black), the presence of LRMP (red), or the presence of IRAG (blue). GVs in the presence of 1 mM cAMP are shown by open symbols. Error bars in this and subsequent panels are SEM, N = 14-22 (See panel D ). Control HCN4 data in panel C are the same as those in panel B. D : Average V 1/2 values for HCN4 in HEK cells in the absence or presence of LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Each individual observation is plotted as a circle. Number of observations for each dataset is given in parentheses. Averages (± SEM) are plotted as squares. E : Average current density in response to a 3 s step to −150 mV of HCN4 in HEK cells in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as D . * indicates P

    Techniques Used: Transferring

    Endogenous LRMP is responsible for the lack of cAMP sensitivity in CHO cells. A-C: Average conductance-voltage relationships for HCN4 in CHO cells in control conditions (black), the presence of CRIPSR Cas9 (grey), the presence of CRISPR Cas9 and gRNAs targeted to CHO LRMP (red), or the presence of CRISPR Cas9 and gRNAs targeted to CHO IRAG (blue). GVs in the presence of 1 mM cAMP are shown by open symbols. Error bars in all panels are SEM, N = 10-15 (See panel D ). HCN4 CRISPR control data for panels A-C are the same. D: Average V 1/2 values for HCN4 in CHO cells in the presence of CRISPR Cas9 and the absence or presence of gRNAs targeted to LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Each individual observation is plotted as a circle and averages are plotted as squares. Number of observations for each dataset are given in parentheses. * indicates P
    Figure Legend Snippet: Endogenous LRMP is responsible for the lack of cAMP sensitivity in CHO cells. A-C: Average conductance-voltage relationships for HCN4 in CHO cells in control conditions (black), the presence of CRIPSR Cas9 (grey), the presence of CRISPR Cas9 and gRNAs targeted to CHO LRMP (red), or the presence of CRISPR Cas9 and gRNAs targeted to CHO IRAG (blue). GVs in the presence of 1 mM cAMP are shown by open symbols. Error bars in all panels are SEM, N = 10-15 (See panel D ). HCN4 CRISPR control data for panels A-C are the same. D: Average V 1/2 values for HCN4 in CHO cells in the presence of CRISPR Cas9 and the absence or presence of gRNAs targeted to LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Each individual observation is plotted as a circle and averages are plotted as squares. Number of observations for each dataset are given in parentheses. * indicates P

    Techniques Used: CRISPR

    LRMP and IRAG are isoform-specific modulators of HCN4. A, C: Average conductance-voltage relationships for HCN1 in control conditions (black), the presence of LRMP (red), or the presence of IRAG (blue). GVs in the presence of 1 mM cAMP are shown by open symbols. Error bars in this and subsequent panels are SEM, N = 5-10 (See panel E ). Control HCN1 data in panel C are the same as those from panel A . Inset: Representative currents of HCN1 elicited with 3 s hyperpolarizations to membrane potentials between −30 mV and −130 mV followed by a 3 s pulse to −50 mV. B, D: Average conductance-voltage relationships for HCN2 in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as A . N = 5-9 (See panel F ). Control HCN2 data in panel D are the same as those from panel B . Inset: Representative currents of HCN2 elicited with 3 s hyperpolarizations to membrane potentials between −50 mV and −150 mV followed by a 3 s pulse to −50 mV. E : Average V 1/2 values for HCN1 in HEK cells in the absence or presence of LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Each individual observation is plotted as a circle and averages are plotted as squares. Number of observations for each dataset are given in parentheses. F : Average V 1/2 values for HCN2 in HEK cells in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as E . * indicates P
    Figure Legend Snippet: LRMP and IRAG are isoform-specific modulators of HCN4. A, C: Average conductance-voltage relationships for HCN1 in control conditions (black), the presence of LRMP (red), or the presence of IRAG (blue). GVs in the presence of 1 mM cAMP are shown by open symbols. Error bars in this and subsequent panels are SEM, N = 5-10 (See panel E ). Control HCN1 data in panel C are the same as those from panel A . Inset: Representative currents of HCN1 elicited with 3 s hyperpolarizations to membrane potentials between −30 mV and −130 mV followed by a 3 s pulse to −50 mV. B, D: Average conductance-voltage relationships for HCN2 in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as A . N = 5-9 (See panel F ). Control HCN2 data in panel D are the same as those from panel B . Inset: Representative currents of HCN2 elicited with 3 s hyperpolarizations to membrane potentials between −50 mV and −150 mV followed by a 3 s pulse to −50 mV. E : Average V 1/2 values for HCN1 in HEK cells in the absence or presence of LRMP (red) or IRAG (blue) and 1 mM cAMP (open). Each individual observation is plotted as a circle and averages are plotted as squares. Number of observations for each dataset are given in parentheses. F : Average V 1/2 values for HCN2 in HEK cells in the absence or presence of LRMP, IRAG, and cAMP using the same color scheme as E . * indicates P

    Techniques Used:

    Identification of LRMP and IRAG as HCN4 interaction partners A : Silver-stained gel showing proteins that were co-immunoprecipitated with HCN4 from CHO and HEK cell extracts. The box at ~70 kDa indicates a potential CHO-specific HCN4 interacting protein that was sequenced using mass-spectroscopy. B : Schematic illustrations of LRMP and IRAG domain structures, showing the relative sizes of the coiled-coil domains, ER transmembrane domains, ER luminal domains, and, in IRAG, the IP3 receptor interaction domain (residues 521-561). C : Relative mRNA abundance of LRMP (red) and IRAG (blue) in CHO and HEK cells as measured by qPCR. Data were normalized to 18S ribosomal RNA abundance and are plotted relative to LRMP abundance in HEK cells. All error bars are SEM. Data are from a minimum of 2 technical replicates of 3 independent biological samples. D : Western blot of anti-Myc staining of extracts of HEK 293 cells stably expressing HCN4 and transiently transfected with Myc-LRMP, Myc-IRAG, or pCDNA3.1. Red boxes show the Myc-LRMP and Myc-IRAG bands. Cell lysates were immunoprecipitated with rabbit anti-HCN4 antibodies. All panels are from the same blot with lanes removed for clarity. Representative of 3 independent blots. WE, Whole extract; S, supernatant; IP, HCN4 immunoprecipitate.
    Figure Legend Snippet: Identification of LRMP and IRAG as HCN4 interaction partners A : Silver-stained gel showing proteins that were co-immunoprecipitated with HCN4 from CHO and HEK cell extracts. The box at ~70 kDa indicates a potential CHO-specific HCN4 interacting protein that was sequenced using mass-spectroscopy. B : Schematic illustrations of LRMP and IRAG domain structures, showing the relative sizes of the coiled-coil domains, ER transmembrane domains, ER luminal domains, and, in IRAG, the IP3 receptor interaction domain (residues 521-561). C : Relative mRNA abundance of LRMP (red) and IRAG (blue) in CHO and HEK cells as measured by qPCR. Data were normalized to 18S ribosomal RNA abundance and are plotted relative to LRMP abundance in HEK cells. All error bars are SEM. Data are from a minimum of 2 technical replicates of 3 independent biological samples. D : Western blot of anti-Myc staining of extracts of HEK 293 cells stably expressing HCN4 and transiently transfected with Myc-LRMP, Myc-IRAG, or pCDNA3.1. Red boxes show the Myc-LRMP and Myc-IRAG bands. Cell lysates were immunoprecipitated with rabbit anti-HCN4 antibodies. All panels are from the same blot with lanes removed for clarity. Representative of 3 independent blots. WE, Whole extract; S, supernatant; IP, HCN4 immunoprecipitate.

    Techniques Used: Staining, Immunoprecipitation, Mass Spectrometry, Real-time Polymerase Chain Reaction, Western Blot, Stable Transfection, Expressing, Transfection

    LRMP IRAG are expressed in mouse sinoatrial node tissue and IRAG is predicted to increase I f during sinoatrial APs. A: Relative mRNA abundance of HCN4 (black), LRMP (red), and IRAG (blue) in mouse sinoatrial node tissue as measured by qPCR. In all cases, data were normalized to 18S ribosomal RNA abundance and are plotted relative to HCN4 abundance in left atrial tissue from the same mice. Error bars are SEM. Data are from a minimum of 4 technical replicates of 3 independent biological samples. B: Western blot of IRAG in lysates from mouse sinoatrial node, left atrium, ventricle, and liver. GAPDH is shown as a loading control. The blot is representative of 3 independent biological samples. C: Schematic of the I f model developed by Verkerk and Wilders ( 46 ). See methods for equations used in the model. D: Simulated voltage-dependence of I f in the wild-type (black lines), IRAG overexpression (blue lines), and IRAG knockout models (red lines) overlaid on experimental data from Larson et al. (black symbols) collected in young mice with basal levels of β-adrenergic stimulation ( 47 ). Inset: Simulated wild-type I f currents during 3 s hyperpolarizing pulses to membrane potentials between −50 mV and −150 mV followed by a 3 s pulse to −50 mV. E: Simulated I f currents in the wild-type (black) , IRAG overexpression (blue), and IRAG knockout models (red) stimulated with a train of action potentials recorded from a mouse sinoatrial node myocyte (top).
    Figure Legend Snippet: LRMP IRAG are expressed in mouse sinoatrial node tissue and IRAG is predicted to increase I f during sinoatrial APs. A: Relative mRNA abundance of HCN4 (black), LRMP (red), and IRAG (blue) in mouse sinoatrial node tissue as measured by qPCR. In all cases, data were normalized to 18S ribosomal RNA abundance and are plotted relative to HCN4 abundance in left atrial tissue from the same mice. Error bars are SEM. Data are from a minimum of 4 technical replicates of 3 independent biological samples. B: Western blot of IRAG in lysates from mouse sinoatrial node, left atrium, ventricle, and liver. GAPDH is shown as a loading control. The blot is representative of 3 independent biological samples. C: Schematic of the I f model developed by Verkerk and Wilders ( 46 ). See methods for equations used in the model. D: Simulated voltage-dependence of I f in the wild-type (black lines), IRAG overexpression (blue lines), and IRAG knockout models (red lines) overlaid on experimental data from Larson et al. (black symbols) collected in young mice with basal levels of β-adrenergic stimulation ( 47 ). Inset: Simulated wild-type I f currents during 3 s hyperpolarizing pulses to membrane potentials between −50 mV and −150 mV followed by a 3 s pulse to −50 mV. E: Simulated I f currents in the wild-type (black) , IRAG overexpression (blue), and IRAG knockout models (red) stimulated with a train of action potentials recorded from a mouse sinoatrial node myocyte (top).

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Western Blot, Over Expression, Knock-Out

    2) Product Images from "Selective Blockade of HCN1/HCN2 Channels as a Potential Pharmacological Strategy Against Pain"

    Article Title: Selective Blockade of HCN1/HCN2 Channels as a Potential Pharmacological Strategy Against Pain

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01252

    Expression and localization of HCN1 (A) , HCN2 (B) , and HCN4 (C) isoforms, and co-expression of HCN1/HCN4 (D) and HCN2/HCN4 (E) isoforms in DRG neurons. Immunofluorescence images of mouse DRG neurons show typical staining of HCN1 and HCN2 (red signal), HCN4 (green signal) and nuclei (blue signal). The white bar on each panel corresponds to 50 μM.
    Figure Legend Snippet: Expression and localization of HCN1 (A) , HCN2 (B) , and HCN4 (C) isoforms, and co-expression of HCN1/HCN4 (D) and HCN2/HCN4 (E) isoforms in DRG neurons. Immunofluorescence images of mouse DRG neurons show typical staining of HCN1 and HCN2 (red signal), HCN4 (green signal) and nuclei (blue signal). The white bar on each panel corresponds to 50 μM.

    Techniques Used: Expressing, Immunofluorescence, Staining

    3) Product Images from "Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits"

    Article Title: Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI99986

    The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats. ( A – D ) LV- Hcn1 -shRNA, LV- Hcn2 -shRNA, LV- Hcn3 -shRNA, and LV- Hcn4 -shRNA effectively downregulated the expression of Hcn1 , Hcn2 , Hcn3 , and Hcn4 mRNAs and proteins ( n = 6 from 6 independent experiments) in the STN. ( E ) LV- Hcn2 -oe upregulated the expression of Hcn2 mRNAs and proteins ( n = 6 from 6 independent experiments). ( F – H ) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior ( F , n = 12), adhesive-removal test ( G , n = 10), and locomotor footprints ( H , n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. *** P
    Figure Legend Snippet: The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats. ( A – D ) LV- Hcn1 -shRNA, LV- Hcn2 -shRNA, LV- Hcn3 -shRNA, and LV- Hcn4 -shRNA effectively downregulated the expression of Hcn1 , Hcn2 , Hcn3 , and Hcn4 mRNAs and proteins ( n = 6 from 6 independent experiments) in the STN. ( E ) LV- Hcn2 -oe upregulated the expression of Hcn2 mRNAs and proteins ( n = 6 from 6 independent experiments). ( F – H ) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior ( F , n = 12), adhesive-removal test ( G , n = 10), and locomotor footprints ( H , n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. *** P

    Techniques Used: shRNA, Expressing, Over Expression, Injection

    4) Product Images from "Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits"

    Article Title: Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI99986

    The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats. ( A – D ) LV- Hcn1 -shRNA, LV- Hcn2 -shRNA, LV- Hcn3 -shRNA, and LV- Hcn4 -shRNA effectively downregulated the expression of Hcn1 , Hcn2 , Hcn3 , and Hcn4 mRNAs and proteins ( n = 6 from 6 independent experiments) in the STN. ( E ) LV- Hcn2 -oe upregulated the expression of Hcn2 mRNAs and proteins ( n = 6 from 6 independent experiments). ( F – H ) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior ( F , n = 12), adhesive-removal test ( G , n = 10), and locomotor footprints ( H , n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. *** P
    Figure Legend Snippet: The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats. ( A – D ) LV- Hcn1 -shRNA, LV- Hcn2 -shRNA, LV- Hcn3 -shRNA, and LV- Hcn4 -shRNA effectively downregulated the expression of Hcn1 , Hcn2 , Hcn3 , and Hcn4 mRNAs and proteins ( n = 6 from 6 independent experiments) in the STN. ( E ) LV- Hcn2 -oe upregulated the expression of Hcn2 mRNAs and proteins ( n = 6 from 6 independent experiments). ( F – H ) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior ( F , n = 12), adhesive-removal test ( G , n = 10), and locomotor footprints ( H , n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. *** P

    Techniques Used: shRNA, Expressing, Over Expression, Injection

    5) Product Images from "Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits"

    Article Title: Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI99986

    The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats. ( A – D ) LV- Hcn1 -shRNA, LV- Hcn2 -shRNA, LV- Hcn3 -shRNA, and LV- Hcn4 -shRNA effectively downregulated the expression of Hcn1 , Hcn2 , Hcn3 , and Hcn4 mRNAs and proteins ( n = 6 from 6 independent experiments) in the STN. ( E ) LV- Hcn2 -oe upregulated the expression of Hcn2 mRNAs and proteins ( n = 6 from 6 independent experiments). ( F – H ) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior ( F , n = 12), adhesive-removal test ( G , n = 10), and locomotor footprints ( H , n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. *** P
    Figure Legend Snippet: The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats. ( A – D ) LV- Hcn1 -shRNA, LV- Hcn2 -shRNA, LV- Hcn3 -shRNA, and LV- Hcn4 -shRNA effectively downregulated the expression of Hcn1 , Hcn2 , Hcn3 , and Hcn4 mRNAs and proteins ( n = 6 from 6 independent experiments) in the STN. ( E ) LV- Hcn2 -oe upregulated the expression of Hcn2 mRNAs and proteins ( n = 6 from 6 independent experiments). ( F – H ) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior ( F , n = 12), adhesive-removal test ( G , n = 10), and locomotor footprints ( H , n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. *** P

    Techniques Used: shRNA, Expressing, Over Expression, Injection

    6) Product Images from "Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits"

    Article Title: Regularizing firing patterns of rat subthalamic neurons ameliorates parkinsonian motor deficits

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI99986

    The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats. ( A – D ) LV- Hcn1 -shRNA, LV- Hcn2 -shRNA, LV- Hcn3 -shRNA, and LV- Hcn4 -shRNA effectively downregulated the expression of Hcn1 , Hcn2 , Hcn3 , and Hcn4 mRNAs and proteins ( n = 6 from 6 independent experiments) in the STN. ( E ) LV- Hcn2 -oe upregulated the expression of Hcn2 mRNAs and proteins ( n = 6 from 6 independent experiments). ( F – H ) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior ( F , n = 12), adhesive-removal test ( G , n = 10), and locomotor footprints ( H , n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. *** P
    Figure Legend Snippet: The HCN2 channel is responsible for the histamine-induced amelioration of motor deficits in PD rats. ( A – D ) LV- Hcn1 -shRNA, LV- Hcn2 -shRNA, LV- Hcn3 -shRNA, and LV- Hcn4 -shRNA effectively downregulated the expression of Hcn1 , Hcn2 , Hcn3 , and Hcn4 mRNAs and proteins ( n = 6 from 6 independent experiments) in the STN. ( E ) LV- Hcn2 -oe upregulated the expression of Hcn2 mRNAs and proteins ( n = 6 from 6 independent experiments). ( F – H ) Effects of downregulation and overexpression of the HCN2 channel in the STN on motor deficits of turning behavior ( F , n = 12), adhesive-removal test ( G , n = 10), and locomotor footprints ( H , n = 10) in PD rats with sham operation, saline injection, and histamine injection. Downregulation of the HCN2 channel significantly increased the apomorphine-induced turnings, prolonged contralesional adhesive-removal time, and shortened bilateral stride length, whereas downregulation of the HCN1, HCN3, or HCN4 channel had no effect on these motor deficits. Only the downregulation of HCN2 rather than the HCN1, HCN3, or HCN4 channel blocked the amelioration of turnings, removal time, and stride length of PD rats induced by microinjection of histamine into the STN. Overexpression of the HCN2 channel in STN not only decreased the turnings, reduced removal time, and enlarged bilateral stride length of PD rats, but also improved the histamine-induced amelioration in these motor behaviors. Data are represented as mean ± SEM. *** P

    Techniques Used: shRNA, Expressing, Over Expression, Injection

    7) Product Images from "Expression of connexin 43, ion channels and Ca2+-handling proteins in rat pulmonary vein cardiomyocytes"

    Article Title: Expression of connexin 43, ion channels and Ca2+-handling proteins in rat pulmonary vein cardiomyocytes

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2016.3766

    Immunohistochemistry of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and connexin 43 (Cx43) expression in transverse sections of the whole heart of rats without arrhythmia. Expression of HCN4 (green) and Cx43 (red) in the (A) SAN, (B) LA, (C) RSPV and (D) LIPV. Scale bar, 20 µm. (E) Masson's trichrome-stained transverse section of the whole heart, boxes represent the locations of A, B, C and D. Scale bar, 5,000 µm. LA, left atrium; SAN, sinoatrial node; RSPV, right superior pulmonary vein; LIPV, left inferior pulmonary vein.
    Figure Legend Snippet: Immunohistochemistry of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and connexin 43 (Cx43) expression in transverse sections of the whole heart of rats without arrhythmia. Expression of HCN4 (green) and Cx43 (red) in the (A) SAN, (B) LA, (C) RSPV and (D) LIPV. Scale bar, 20 µm. (E) Masson's trichrome-stained transverse section of the whole heart, boxes represent the locations of A, B, C and D. Scale bar, 5,000 µm. LA, left atrium; SAN, sinoatrial node; RSPV, right superior pulmonary vein; LIPV, left inferior pulmonary vein.

    Techniques Used: Immunohistochemistry, Expressing, Staining

    8) Product Images from "Expression of connexin 43, ion channels and Ca2+-handling proteins in rat pulmonary vein cardiomyocytes"

    Article Title: Expression of connexin 43, ion channels and Ca2+-handling proteins in rat pulmonary vein cardiomyocytes

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2016.3766

    Immunohistochemistry of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and connexin 43 (Cx43) expression in transverse sections of the whole heart of rats without arrhythmia. Expression of HCN4 (green) and Cx43 (red) in the (A) SAN, (B) LA, (C) RSPV and (D) LIPV. Scale bar, 20 µm. (E) Masson's trichrome-stained transverse section of the whole heart, boxes represent the locations of A, B, C and D. Scale bar, 5,000 µm. LA, left atrium; SAN, sinoatrial node; RSPV, right superior pulmonary vein; LIPV, left inferior pulmonary vein.
    Figure Legend Snippet: Immunohistochemistry of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and connexin 43 (Cx43) expression in transverse sections of the whole heart of rats without arrhythmia. Expression of HCN4 (green) and Cx43 (red) in the (A) SAN, (B) LA, (C) RSPV and (D) LIPV. Scale bar, 20 µm. (E) Masson's trichrome-stained transverse section of the whole heart, boxes represent the locations of A, B, C and D. Scale bar, 5,000 µm. LA, left atrium; SAN, sinoatrial node; RSPV, right superior pulmonary vein; LIPV, left inferior pulmonary vein.

    Techniques Used: Immunohistochemistry, Expressing, Staining

    9) Product Images from "Gsg1, Trnp1, and Tmem215 Mark Subpopulations of Bipolar Interneurons in the Mouse Retina"

    Article Title: Gsg1, Trnp1, and Tmem215 Mark Subpopulations of Bipolar Interneurons in the Mouse Retina

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.16-19767

    Tmem215 marks subsets of bipolar and amacrine cells. Adult Tmem215-LacZ heterozygous mice stained for β-gal ( green ) and cell-type specific markers ( red / gray ). ( A – A' ) Section stained with Scgn ( gray ) and PKCα ( red ). A large fraction of β-gal+ cells coexpress Scgn ( arrows , insets ), but none overlap with PKCα ( arrowheads , insets ). ( B ) Costaining with Scgn ( gray ) and Isl1/2 ( red ) to mark ON bipolar cells. A subset of β-gal+ cells coexpress Isl1/2 ( arrows , blue insets ). Other β-gal+ cells coexpress only Scgn ( arrowheads ), marking them as cone OFF bipolars. Thus, Tmem215-LacZ marks both ON and OFF cone bipolar cells. ( C – C' ) Costaining with Isl1/2 ( gray ) and Vsx1 ( red ), which mark types 1, 2, and 7 cone bipolars. A subset of β-gal+ cells coexpress Vsx1 and Isl1/2 ( arrows , insets ), marking them as type 7 cone ON bipolars. However, not all type 7 cone bipolars were β-gal+ ( magenta arrowheads , insets ). Some β-gal+ cells expressed Isl1/2, but not Vsx1 ( arrowheads , insets ). Of the β-gal+ cells that did not express Isl1/2, none coexpressed Vsx1 ( asterisks ). This argues that types 1 and 2 cone bipolars are not Tmem215+. Isl1/2+ amacrine cells do not coexpress β-gal. ( D ) A subset of β-gal+ cells coexpress Cabp5 ( red , arrows , insets ), which marks types 3 and 5 cone bipolars. ( E ) β-gal costaining with HCN4 to mark type 3a cone OFF bipolars. Most HCN4+ bipolar cells coexpress β-gal ( arrows , insets ), though some HCN4+ cells in the inner INL lack β-gal staining ( arrowheads ). ( F ) β-gal expression ( arrowheads , insets ) does not overlap with PKARIIβ ( red ), a marker of type 3b cone bipolars. ( G ) Type 2 cone OFF bipolars marked with Bhlhb5 did not express β-gal ( arrowheads , insets ). We did not see β-gal overlap with the type 4 cone OFF bipolar marker Csen (data not shown). ( H – H' ) Retinas stained with the amacrine markers GlyT1 ( red , glycinergic) and GAD65/67 ( gray , GABAergic). Roughly equal fractions coexpress GlyT1 ( arrows , insets ) and GAD65/67 ( arrowheads , insets ). There are no β-gal+ displaced amacrine cells seen. ( I ) A subset of the β-gal+ amacrine cells ( arrowheads , insets ) coexpress Ap2α ( red ) ( arrows , insets ). Scale bars : 50 μm for panels and 10 μm for insets. ( J ) Quantification of β-gal+ cells. The left panel shows the percentage of β-gal+ cells that coexpress a cell type–specific marker. There are approximately 9 β-gal+ bipolar cells (Otx2+) for every amacrine cell (Pax6+). The right panel shows the fraction of a cell type–specific marker population that coexpresses β-gal+. Error bars show SD.
    Figure Legend Snippet: Tmem215 marks subsets of bipolar and amacrine cells. Adult Tmem215-LacZ heterozygous mice stained for β-gal ( green ) and cell-type specific markers ( red / gray ). ( A – A' ) Section stained with Scgn ( gray ) and PKCα ( red ). A large fraction of β-gal+ cells coexpress Scgn ( arrows , insets ), but none overlap with PKCα ( arrowheads , insets ). ( B ) Costaining with Scgn ( gray ) and Isl1/2 ( red ) to mark ON bipolar cells. A subset of β-gal+ cells coexpress Isl1/2 ( arrows , blue insets ). Other β-gal+ cells coexpress only Scgn ( arrowheads ), marking them as cone OFF bipolars. Thus, Tmem215-LacZ marks both ON and OFF cone bipolar cells. ( C – C' ) Costaining with Isl1/2 ( gray ) and Vsx1 ( red ), which mark types 1, 2, and 7 cone bipolars. A subset of β-gal+ cells coexpress Vsx1 and Isl1/2 ( arrows , insets ), marking them as type 7 cone ON bipolars. However, not all type 7 cone bipolars were β-gal+ ( magenta arrowheads , insets ). Some β-gal+ cells expressed Isl1/2, but not Vsx1 ( arrowheads , insets ). Of the β-gal+ cells that did not express Isl1/2, none coexpressed Vsx1 ( asterisks ). This argues that types 1 and 2 cone bipolars are not Tmem215+. Isl1/2+ amacrine cells do not coexpress β-gal. ( D ) A subset of β-gal+ cells coexpress Cabp5 ( red , arrows , insets ), which marks types 3 and 5 cone bipolars. ( E ) β-gal costaining with HCN4 to mark type 3a cone OFF bipolars. Most HCN4+ bipolar cells coexpress β-gal ( arrows , insets ), though some HCN4+ cells in the inner INL lack β-gal staining ( arrowheads ). ( F ) β-gal expression ( arrowheads , insets ) does not overlap with PKARIIβ ( red ), a marker of type 3b cone bipolars. ( G ) Type 2 cone OFF bipolars marked with Bhlhb5 did not express β-gal ( arrowheads , insets ). We did not see β-gal overlap with the type 4 cone OFF bipolar marker Csen (data not shown). ( H – H' ) Retinas stained with the amacrine markers GlyT1 ( red , glycinergic) and GAD65/67 ( gray , GABAergic). Roughly equal fractions coexpress GlyT1 ( arrows , insets ) and GAD65/67 ( arrowheads , insets ). There are no β-gal+ displaced amacrine cells seen. ( I ) A subset of the β-gal+ amacrine cells ( arrowheads , insets ) coexpress Ap2α ( red ) ( arrows , insets ). Scale bars : 50 μm for panels and 10 μm for insets. ( J ) Quantification of β-gal+ cells. The left panel shows the percentage of β-gal+ cells that coexpress a cell type–specific marker. There are approximately 9 β-gal+ bipolar cells (Otx2+) for every amacrine cell (Pax6+). The right panel shows the fraction of a cell type–specific marker population that coexpresses β-gal+. Error bars show SD.

    Techniques Used: Mouse Assay, Staining, Expressing, Marker

    10) Product Images from "Developmental Localization of Adhesion and Scaffolding Proteins at the Cone Synapse"

    Article Title: Developmental Localization of Adhesion and Scaffolding Proteins at the Cone Synapse

    Journal: Gene expression patterns : GEP

    doi: 10.1016/j.gep.2014.07.003

    Localization of γ-protocadherin A - E , Sections of retina were labeled with PNA and antibodies to PSD95 and γ-protocadherin (N=3 at each age). A , Diffuse γ-protocadherin staining is observed in the developing OPL at postnatal day 6. B , By postnatal day 8, puncta of γ-protocadherin immunoreactivity that do not overlap with PSD95 were observed in the developing OPL. C , At postnatal day 10, concentrations of γ-protocadherin puncta were observed INL-proximal to PNA staining. Some γ-protocadherin staining was also observed in the soma of cells within the INL. D and E , After postnatal day 10, γ-protocadherin staining was observed concentrated INL-proximal to PNA staining, a pattern that persists in the adult retina. F , γ-protocadherin staining in the macaque retina was similar to what was observed in mouse, with a concentration of γ-protocadherin puncta INL-proximal to and overlapping with PNA staining. G , Some overlap between γ-protocadherin staining and calbindin was observed. H , γ-protocadherin staining proximal to PNA staining did not overlap with GS staining. I , γ-protocadherin staining overlapped with bipolar type specific markers, such as HCN4, which is expressed in type 3a OFF cone bipolar cells. The scale bar in ( I ) is equivalent to 31.9 μm in A - F (insets are 8.5 μm) and 21.3 μm in G - I .
    Figure Legend Snippet: Localization of γ-protocadherin A - E , Sections of retina were labeled with PNA and antibodies to PSD95 and γ-protocadherin (N=3 at each age). A , Diffuse γ-protocadherin staining is observed in the developing OPL at postnatal day 6. B , By postnatal day 8, puncta of γ-protocadherin immunoreactivity that do not overlap with PSD95 were observed in the developing OPL. C , At postnatal day 10, concentrations of γ-protocadherin puncta were observed INL-proximal to PNA staining. Some γ-protocadherin staining was also observed in the soma of cells within the INL. D and E , After postnatal day 10, γ-protocadherin staining was observed concentrated INL-proximal to PNA staining, a pattern that persists in the adult retina. F , γ-protocadherin staining in the macaque retina was similar to what was observed in mouse, with a concentration of γ-protocadherin puncta INL-proximal to and overlapping with PNA staining. G , Some overlap between γ-protocadherin staining and calbindin was observed. H , γ-protocadherin staining proximal to PNA staining did not overlap with GS staining. I , γ-protocadherin staining overlapped with bipolar type specific markers, such as HCN4, which is expressed in type 3a OFF cone bipolar cells. The scale bar in ( I ) is equivalent to 31.9 μm in A - F (insets are 8.5 μm) and 21.3 μm in G - I .

    Techniques Used: Labeling, Staining, Concentration Assay

    11) Product Images from "A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation"

    Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

    Journal: Heart rhythm : the official journal of the Heart Rhythm Society

    doi: 10.1016/j.hrthm.2014.03.002

    The p.Pro257Ser mutant channel does not produce a measurable current and does not express on the cell membrane A, Currents were elicited from a holding current of -35 mV to a test pulse of -150 mV (fully-activated voltage) for 4 seconds and returned back to the holding current. The wild type HCN4 channel produced a current and the p.Pro257Ser mutant did not. B, confocal micrographs of wild type HCN4 and p.Pro257Ser channels expressed in CHO cells. The cells were stained with rabbit anti-HCN4 antibody (green) and DAPI (blue). The wild type HCN4 channel is expressed on the cell membrane and in the cytoplasm, whereas the p.Pro257Ser mutant channel is restricted to the cytoplasm. The scale bar denotes 50μm.
    Figure Legend Snippet: The p.Pro257Ser mutant channel does not produce a measurable current and does not express on the cell membrane A, Currents were elicited from a holding current of -35 mV to a test pulse of -150 mV (fully-activated voltage) for 4 seconds and returned back to the holding current. The wild type HCN4 channel produced a current and the p.Pro257Ser mutant did not. B, confocal micrographs of wild type HCN4 and p.Pro257Ser channels expressed in CHO cells. The cells were stained with rabbit anti-HCN4 antibody (green) and DAPI (blue). The wild type HCN4 channel is expressed on the cell membrane and in the cytoplasm, whereas the p.Pro257Ser mutant channel is restricted to the cytoplasm. The scale bar denotes 50μm.

    Techniques Used: Mutagenesis, Produced, Staining

    Co-expression of the wild type HCN4 channel the p.Pro257Ser mutant channel produce currents that are not functionally different from wild type A, current recordings of wild type HCN4 (2 μg) and wild type HCN4 (1 μg)+p.Pro257Ser (1 μg). B, plot of current density (pA/pF) measured at -150 mV for wild type HCN4 and wild type HCN4+p.Pro257Ser. C, plots of activation curves for wild type HCN4 and wild type HCN4+p.Pro257Ser. The number in parentheses represents the number of cells. D, confocal micrographs of co-expressed wild type HCN4 and p.Pro257Ser constructs tagged with unique C-terminal epitopes in CHO cells (see methods); i) wild type HCN4-myc (green)+ wild type HCN4-V5 (red) and ii) wild type HCN4-myc (green)+p.Pro257Ser-V5(red). Co-expressed wild type HCN4-myc and wild type HCN4-V5 channels both traffick and are distributed together on cell membrane. ii) wild type HCN4-myc +p.Pro257Ser-V5 images show the wild type HCN4-myc channel expressed on the cell membrane and the p.Pro257Ser-V5 channel distributed in the cytoplasm and not on the cell membrane. Cells were also stained with DAPI (blue) to visualize the nucleus which is shown in the merged images. The scale bar denotes 50μm.
    Figure Legend Snippet: Co-expression of the wild type HCN4 channel the p.Pro257Ser mutant channel produce currents that are not functionally different from wild type A, current recordings of wild type HCN4 (2 μg) and wild type HCN4 (1 μg)+p.Pro257Ser (1 μg). B, plot of current density (pA/pF) measured at -150 mV for wild type HCN4 and wild type HCN4+p.Pro257Ser. C, plots of activation curves for wild type HCN4 and wild type HCN4+p.Pro257Ser. The number in parentheses represents the number of cells. D, confocal micrographs of co-expressed wild type HCN4 and p.Pro257Ser constructs tagged with unique C-terminal epitopes in CHO cells (see methods); i) wild type HCN4-myc (green)+ wild type HCN4-V5 (red) and ii) wild type HCN4-myc (green)+p.Pro257Ser-V5(red). Co-expressed wild type HCN4-myc and wild type HCN4-V5 channels both traffick and are distributed together on cell membrane. ii) wild type HCN4-myc +p.Pro257Ser-V5 images show the wild type HCN4-myc channel expressed on the cell membrane and the p.Pro257Ser-V5 channel distributed in the cytoplasm and not on the cell membrane. Cells were also stained with DAPI (blue) to visualize the nucleus which is shown in the merged images. The scale bar denotes 50μm.

    Techniques Used: Expressing, Mutagenesis, Activation Assay, Construct, Staining

    ECG recordings from AF-22 who carries the HCN4 trafficking-defective p.Pro257Ser mutation Surface ECG of leads II and V1 from AF-22. AF-22 presents with the absence of p-waves indicative of AF and conduction pauses at the time of enrollment and remains in AF 12 years later.
    Figure Legend Snippet: ECG recordings from AF-22 who carries the HCN4 trafficking-defective p.Pro257Ser mutation Surface ECG of leads II and V1 from AF-22. AF-22 presents with the absence of p-waves indicative of AF and conduction pauses at the time of enrollment and remains in AF 12 years later.

    Techniques Used: Mutagenesis

    Location of novel HCN4 coding variants in early-onset AF cases and referents A B, illustration of a HCN4 -subunit with the cytoplasmic NH2- and COOH-terminus, six transmembrane segments (S1–S6), including the S4 voltage sensor (‘+’ sign denotes amino acid residues with positive charge), the pore loop between S5 and S6, and the C-linker (CL) with the cyclic-nucleotide binding domain (CNBD). The red (seven) and blue (three) circles denote the location of the novel variants identified in the early-onset AF cases and referents, respectively.
    Figure Legend Snippet: Location of novel HCN4 coding variants in early-onset AF cases and referents A B, illustration of a HCN4 -subunit with the cytoplasmic NH2- and COOH-terminus, six transmembrane segments (S1–S6), including the S4 voltage sensor (‘+’ sign denotes amino acid residues with positive charge), the pore loop between S5 and S6, and the C-linker (CL) with the cyclic-nucleotide binding domain (CNBD). The red (seven) and blue (three) circles denote the location of the novel variants identified in the early-onset AF cases and referents, respectively.

    Techniques Used: Binding Assay

    12) Product Images from "Cre-mediated recombination efficiency and transgene expression patterns of three retinal bipolar cell-expressing Cre transgenic mouse lines"

    Article Title: Cre-mediated recombination efficiency and transgene expression patterns of three retinal bipolar cell-expressing Cre transgenic mouse lines

    Journal: Molecular Vision

    doi:

    The tdTomato-expressing retinal bipolar cells in the 5-HTR2a-cre mouse line are co-labeled with antibodies specific to type 4 and type 3b cone bipolar cells, and rod bipolar cells. A – C : In a retinal vertical section, the tdTomato-expressing retina ( A ) was immunostained for calsenilin ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells are marked with stars. D – F : In a retinal whole mount with the focal plane at the distal portion of the inner nuclear layer (INL), the tdTomato-expressing retina ( D ) was immunostained for calsenilin ( E ). The overlay of D and E is shown in F . The majority of the tomato-expressing cells in the distal portion of the INL are calsenilin-positive. The tdTomato-expressing cells that do not show calsenilin staining are marked with arrowheads. The tdTomato-expressing retina was immunostained for PKCα in a retinal vertical section ( G–I ). The double-positive bipolar cells are marked with arrows in the somata and arrowheads pointing to the axon terminals. J – L : The tdTomato-expressing retina was immunostained for PKARIIβ. Two double-positive cells are marked with stars. The tdTomato-expressing retinal bipolar cells were not labeled by antibodies for HCN4 ( M – O ) and Syt2 ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
    Figure Legend Snippet: The tdTomato-expressing retinal bipolar cells in the 5-HTR2a-cre mouse line are co-labeled with antibodies specific to type 4 and type 3b cone bipolar cells, and rod bipolar cells. A – C : In a retinal vertical section, the tdTomato-expressing retina ( A ) was immunostained for calsenilin ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells are marked with stars. D – F : In a retinal whole mount with the focal plane at the distal portion of the inner nuclear layer (INL), the tdTomato-expressing retina ( D ) was immunostained for calsenilin ( E ). The overlay of D and E is shown in F . The majority of the tomato-expressing cells in the distal portion of the INL are calsenilin-positive. The tdTomato-expressing cells that do not show calsenilin staining are marked with arrowheads. The tdTomato-expressing retina was immunostained for PKCα in a retinal vertical section ( G–I ). The double-positive bipolar cells are marked with arrows in the somata and arrowheads pointing to the axon terminals. J – L : The tdTomato-expressing retina was immunostained for PKARIIβ. Two double-positive cells are marked with stars. The tdTomato-expressing retinal bipolar cells were not labeled by antibodies for HCN4 ( M – O ) and Syt2 ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Techniques Used: Expressing, Labeling, Staining

    The tdTomato-expressing retinal bipolar cells in the Pcp2-cre mouse line are co-labeled with antibodies specific to rod bipolar cells, type 2 and 6 cone bipolar cells. A – C : In retinal vertical sections, the tdTomato-expressing retina ( A ) was immunostained for PKCα ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells were marked with stars in the somata and with arrowheads pointing at the axon terminals. D–F : In the retinal whole mount with the focal plane in the INL, colabeling with tdTomato and PKCα. PKCα-negative tdTomato-expressing cells are marked with arrows. G–I : The tdTomato-expressing retina was immunostained for Syt2. The double-positive bipolar cells with axon terminals stratified at the distal portion of the IPL (type 2 bipolar cells) are marked with white stars in the somata and with white arrowheads pointing at the axon terminals. The double-positive bipolar cells with axon terminals stratified in the proximal portion of the IPL (type 6 bipolar cells) are marked with yellow arrows at the somata and yellow arrowheads at the axon terminals. A tdTomato-expressing bipolar cell with their axon terminals stratified slightly distal to Syt2-positive cells is marked with a blue arrowhead ( I ). The tdTomato-expressing retinal bipolar cells were not found to be labeled by PKARIIβ ( J – L ), HCN4 ( M – O ), and calsenilin ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
    Figure Legend Snippet: The tdTomato-expressing retinal bipolar cells in the Pcp2-cre mouse line are co-labeled with antibodies specific to rod bipolar cells, type 2 and 6 cone bipolar cells. A – C : In retinal vertical sections, the tdTomato-expressing retina ( A ) was immunostained for PKCα ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells were marked with stars in the somata and with arrowheads pointing at the axon terminals. D–F : In the retinal whole mount with the focal plane in the INL, colabeling with tdTomato and PKCα. PKCα-negative tdTomato-expressing cells are marked with arrows. G–I : The tdTomato-expressing retina was immunostained for Syt2. The double-positive bipolar cells with axon terminals stratified at the distal portion of the IPL (type 2 bipolar cells) are marked with white stars in the somata and with white arrowheads pointing at the axon terminals. The double-positive bipolar cells with axon terminals stratified in the proximal portion of the IPL (type 6 bipolar cells) are marked with yellow arrows at the somata and yellow arrowheads at the axon terminals. A tdTomato-expressing bipolar cell with their axon terminals stratified slightly distal to Syt2-positive cells is marked with a blue arrowhead ( I ). The tdTomato-expressing retinal bipolar cells were not found to be labeled by PKARIIβ ( J – L ), HCN4 ( M – O ), and calsenilin ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Techniques Used: Expressing, Labeling

    13) Product Images from "Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains"

    Article Title: Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains

    Journal: Neurobiology of disease

    doi: 10.1016/j.nbd.2011.08.032

    Immunohistochemical characterization of HCN channel localization in dLGN. ( A ) Specific antibodies directed against HCN1 (1 st column), HCN2 (2 nd column), HCN3 (3 rd column), and HCN4 (4 th column) were applied in WAG/Rij (upper row) and ACI (lower row) rats.
    Figure Legend Snippet: Immunohistochemical characterization of HCN channel localization in dLGN. ( A ) Specific antibodies directed against HCN1 (1 st column), HCN2 (2 nd column), HCN3 (3 rd column), and HCN4 (4 th column) were applied in WAG/Rij (upper row) and ACI (lower row) rats.

    Techniques Used: Immunohistochemistry

    Quantitative analyses of postnatal protein expression of HCN channels in dLGN tissue. HCN1 ( A ), HCN3 ( B ), HCN2 ( C ), and HCN4 ( D ) were detected by means of Western blotting (A = ACI, gray bars; W = WAG/Rij, black bars). Data from 4 independent protein
    Figure Legend Snippet: Quantitative analyses of postnatal protein expression of HCN channels in dLGN tissue. HCN1 ( A ), HCN3 ( B ), HCN2 ( C ), and HCN4 ( D ) were detected by means of Western blotting (A = ACI, gray bars; W = WAG/Rij, black bars). Data from 4 independent protein

    Techniques Used: Expressing, Western Blot

    14) Product Images from "Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains"

    Article Title: Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains

    Journal: Neurobiology of disease

    doi: 10.1016/j.nbd.2011.08.032

    Immunohistochemical characterization of HCN channel localization in dLGN. ( A ) Specific antibodies directed against HCN1 (1 st column), HCN2 (2 nd column), HCN3 (3 rd column), and HCN4 (4 th column) were applied in WAG/Rij (upper row) and ACI (lower row) rats.
    Figure Legend Snippet: Immunohistochemical characterization of HCN channel localization in dLGN. ( A ) Specific antibodies directed against HCN1 (1 st column), HCN2 (2 nd column), HCN3 (3 rd column), and HCN4 (4 th column) were applied in WAG/Rij (upper row) and ACI (lower row) rats.

    Techniques Used: Immunohistochemistry

    Quantitative analyses of postnatal protein expression of HCN channels in dLGN tissue. HCN1 ( A ), HCN3 ( B ), HCN2 ( C ), and HCN4 ( D ) were detected by means of Western blotting (A = ACI, gray bars; W = WAG/Rij, black bars). Data from 4 independent protein
    Figure Legend Snippet: Quantitative analyses of postnatal protein expression of HCN channels in dLGN tissue. HCN1 ( A ), HCN3 ( B ), HCN2 ( C ), and HCN4 ( D ) were detected by means of Western blotting (A = ACI, gray bars; W = WAG/Rij, black bars). Data from 4 independent protein

    Techniques Used: Expressing, Western Blot

    15) Product Images from "Postnatal expression pattern of HCN channel isoforms in thalamic neurons: relationship to maturation of thalamocortical oscillations"

    Article Title: Postnatal expression pattern of HCN channel isoforms in thalamic neurons: relationship to maturation of thalamocortical oscillations

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.0689-09.2009

    Quantitative analyses of postnatal mRNA and protein expression of HCN channels. ( A ) HCN1, HCN2, and HCN4 mRNA levels in dLGN were obtained using radioactively labeled in situ hybridization probes (n = 3–6 / group). Significance is indicated for
    Figure Legend Snippet: Quantitative analyses of postnatal mRNA and protein expression of HCN channels. ( A ) HCN1, HCN2, and HCN4 mRNA levels in dLGN were obtained using radioactively labeled in situ hybridization probes (n = 3–6 / group). Significance is indicated for

    Techniques Used: Expressing, Labeling, In Situ Hybridization

    Immunohistochemical characterization of HCN channel localization. Specific antibodies directed against HCN1 ( A ), HCN2 ( B ) and HCN4 ( C ) were applied. All isoforms were detected in the dorsal lateral geniculate nucleus (dLGN) and immunoreactivity was stronger
    Figure Legend Snippet: Immunohistochemical characterization of HCN channel localization. Specific antibodies directed against HCN1 ( A ), HCN2 ( B ) and HCN4 ( C ) were applied. All isoforms were detected in the dorsal lateral geniculate nucleus (dLGN) and immunoreactivity was stronger

    Techniques Used: Immunohistochemistry

    16) Product Images from "CHARACTERIZATION OF GREEN FLUORESCENT PROTEIN-EXPRESSING RETINAL CONE BIPOLAR CELLS IN A 5-HYDROXYTRYPTAMINE RECEPTOR 2a TRANSGENIC MOUSE LINE"

    Article Title: CHARACTERIZATION OF GREEN FLUORESCENT PROTEIN-EXPRESSING RETINAL CONE BIPOLAR CELLS IN A 5-HYDROXYTRYPTAMINE RECEPTOR 2a TRANSGENIC MOUSE LINE

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2009.07.002

    Double labeling using GFP and retinal bipolar cell-specific antibodies. A-C, A retinal vertical section was immunostained for GFP (A) and recoverin (B). The overlay of A and B (C). Double immunostaining for GFP and HCN4 (D-F), for GFP and PKARIIβ
    Figure Legend Snippet: Double labeling using GFP and retinal bipolar cell-specific antibodies. A-C, A retinal vertical section was immunostained for GFP (A) and recoverin (B). The overlay of A and B (C). Double immunostaining for GFP and HCN4 (D-F), for GFP and PKARIIβ

    Techniques Used: Labeling, Double Immunostaining

    17) Product Images from "Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking"

    Article Title: Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2004.068643

    Labelling of Cx43, desmoplakin, HCN4, Na v 1.5 and Na v 1.1 in SA node sections A , schematic diagram of a section. B , double labelling of Cx43 (green) and desmoplakin (DP; red). C , labelling of HCN4. D , labelling of Na v 1.5. E , labelling of Na v 1.1. Table: summary of the expression of the different proteins in the regions shown in A . Scale bars, 200 μm.
    Figure Legend Snippet: Labelling of Cx43, desmoplakin, HCN4, Na v 1.5 and Na v 1.1 in SA node sections A , schematic diagram of a section. B , double labelling of Cx43 (green) and desmoplakin (DP; red). C , labelling of HCN4. D , labelling of Na v 1.5. E , labelling of Na v 1.1. Table: summary of the expression of the different proteins in the regions shown in A . Scale bars, 200 μm.

    Techniques Used: Expressing

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    Alomone Labs cyclic nucleotide gate cation channels hcn4
    A dual immunolabeled <t>HCN4</t> (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Stacked confocal images and reconstructed front view of optically sliced z-stack images of CX43 at a depth of 35 μm from endocardium. Gap junctions are color coded by depth and plotted within the z-stacks reconstructed from optical slices. Note the alignment of immunolabeling at any given depth. Confocal images were acquired with a 40x oil immersion. Panel B: Stacked confocal images and reconstructed side view of optically sliced z-stack images of HCN4 + /F-actin − /(CX43) − cells (red) and HCN4 − /F-actin + /(CX43) + cells (cyan) at a depth of 40 μm and 50 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective Panel C: Optical slice shows HCN4 + /F-actin − /(CX43) − cells (red) adjacent to HCN4 − /F-actin + /(CX43) + cells (cyan). CX43 protein (green) is expressed only in cyan cells.
    Cyclic Nucleotide Gate Cation Channels Hcn4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs polyclonal rabbit rb anti hcn2
    Alteration in HCN channel expression in thalamus following general demyelination. ( A , B ) Immunofluorescence staining of the VB complex (horizontal thalamic sections, 40 μm) comparing the expression of <t>HCN4</t> (A, in red, <t>rb-anti-HCN4,</t> 1:200, Alomone) and <t>HCN2</t> (B, in red, rb-anti-HCN4, 1:200, Alomone) channels between control C3H/HeJ and Day1. The purified ms-anti- neurofilament antibody (SMI312, pan axonal, 1:200, BioLegend) was used as an axonal marker (SMI312, in green). Cell nuclei were stained with DAPI (in blue). ( C , D ) Bar graphs comparing the intensity of the fluorescence signal (using integrated fluorescence intensity values) for SMI312 (C and D upper traces) and HCN4 and HCN2 (lower traces) between the control C3H/HeJ and Day1. ( E ) Immunofluorescence staining of VB in control C3H/HeJ and Day1 with antibodies against TRIP8b (ms-anti-(constant) TRIP8b, 1:50, NeuroMab, in green) and phosphorylated TRIP8b (rb-α-pS237 antibody, 1:100, YenZym). ( F ) Representative bar graph comparing the intensity of the fluorescence signal for total TRIP8b and pS237 between the two groups indicating a significant reduction for phosphorylated TRIP8b, pS237, on Day1. Scale bars indicate 100 μm. VPL, VPM, and TRN stand for ventral-posterior medial, ventral-posterior lateral, and thalamic reticular nucleus, respectively.
    Polyclonal Rabbit Rb Anti Hcn2, 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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    Image Search Results


    A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Stacked confocal images and reconstructed front view of optically sliced z-stack images of CX43 at a depth of 35 μm from endocardium. Gap junctions are color coded by depth and plotted within the z-stacks reconstructed from optical slices. Note the alignment of immunolabeling at any given depth. Confocal images were acquired with a 40x oil immersion. Panel B: Stacked confocal images and reconstructed side view of optically sliced z-stack images of HCN4 + /F-actin − /(CX43) − cells (red) and HCN4 − /F-actin + /(CX43) + cells (cyan) at a depth of 40 μm and 50 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective Panel C: Optical slice shows HCN4 + /F-actin − /(CX43) − cells (red) adjacent to HCN4 − /F-actin + /(CX43) + cells (cyan). CX43 protein (green) is expressed only in cyan cells.

    Journal: JACC. Clinical electrophysiology

    Article Title: Synchronized cardiac impulses emerge from heterogeneous local calcium signals within and among cells of pacemaker tissue

    doi: 10.1016/j.jacep.2020.06.022

    Figure Lengend Snippet: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Stacked confocal images and reconstructed front view of optically sliced z-stack images of CX43 at a depth of 35 μm from endocardium. Gap junctions are color coded by depth and plotted within the z-stacks reconstructed from optical slices. Note the alignment of immunolabeling at any given depth. Confocal images were acquired with a 40x oil immersion. Panel B: Stacked confocal images and reconstructed side view of optically sliced z-stack images of HCN4 + /F-actin − /(CX43) − cells (red) and HCN4 − /F-actin + /(CX43) + cells (cyan) at a depth of 40 μm and 50 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective Panel C: Optical slice shows HCN4 + /F-actin − /(CX43) − cells (red) adjacent to HCN4 − /F-actin + /(CX43) + cells (cyan). CX43 protein (green) is expressed only in cyan cells.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques: Immunolabeling

    Panels A and B: Optical slices (1 μm thick) of HCN4 immunoreactive cells at a depth of 30 μm and 60 μm from the endocardial surface. Panel C: a 3D reconstruction of 70 stacked HCN4 images (optically sliced via confocal microscope) of an immunolabeled whole-mount SAN preparation demonstrating the distribution of HCN4 immunoreactive cells within a depth of 70 μM from the endocardium. Tissue depth from the endocardial site is color coded on the right side of the panel.

    Journal: JACC. Clinical electrophysiology

    Article Title: Synchronized cardiac impulses emerge from heterogeneous local calcium signals within and among cells of pacemaker tissue

    doi: 10.1016/j.jacep.2020.06.022

    Figure Lengend Snippet: Panels A and B: Optical slices (1 μm thick) of HCN4 immunoreactive cells at a depth of 30 μm and 60 μm from the endocardial surface. Panel C: a 3D reconstruction of 70 stacked HCN4 images (optically sliced via confocal microscope) of an immunolabeled whole-mount SAN preparation demonstrating the distribution of HCN4 immunoreactive cells within a depth of 70 μM from the endocardium. Tissue depth from the endocardial site is color coded on the right side of the panel.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques: Microscopy, Immunolabeling

    Panel A: An Image of a whole mount SAN preparation at low (2.5x) optical magnification, demonstrating the distribution of HCN4 (red color) immunoreactive and F-actin (cyan color) labelled cells. The merged images between HCN4 and F-actin is shown in both panels. Panel B: Tiled Image of the HCN4 + /F-actin − cell meshwork (red) intertwined with the HCN4 − /F-actin + cell network (cyan) reconstructed from 4 images recorded via 10x water immersion objective within the red box in panel A.

    Journal: JACC. Clinical electrophysiology

    Article Title: Synchronized cardiac impulses emerge from heterogeneous local calcium signals within and among cells of pacemaker tissue

    doi: 10.1016/j.jacep.2020.06.022

    Figure Lengend Snippet: Panel A: An Image of a whole mount SAN preparation at low (2.5x) optical magnification, demonstrating the distribution of HCN4 (red color) immunoreactive and F-actin (cyan color) labelled cells. The merged images between HCN4 and F-actin is shown in both panels. Panel B: Tiled Image of the HCN4 + /F-actin − cell meshwork (red) intertwined with the HCN4 − /F-actin + cell network (cyan) reconstructed from 4 images recorded via 10x water immersion objective within the red box in panel A.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques:

    Panel A: A dual immunolabeled HCN4 (red) and CX43 (green) whole mount SAN image at low optical magnification (2.5x). Merged (CX43 and HCN4) immunoreactivity is shown in all three panels. Panel B: Image within the ROI in panel A reconstructed from 4 tile images of the HCN4 + /CX43 − meshwork (red) intertwined with HCN4 − /(CX43) + network (green) taken with 10x water immersion objective. Panels C: Confocal images from the area within the ROI in panel B showing: HCN4 + cells that do not express CX43 (upper image); intertwining areas between HCN4 + /(Cx43) − meshwork (red color), and penetrating HCN4-/ CX43 + cells outlined by green dots corresponding to CX43 protein on the cell membranes (middle and lower panel). Note that HCN4 expressing cells in all three images do not express CX43.

    Journal: JACC. Clinical electrophysiology

    Article Title: Synchronized cardiac impulses emerge from heterogeneous local calcium signals within and among cells of pacemaker tissue

    doi: 10.1016/j.jacep.2020.06.022

    Figure Lengend Snippet: Panel A: A dual immunolabeled HCN4 (red) and CX43 (green) whole mount SAN image at low optical magnification (2.5x). Merged (CX43 and HCN4) immunoreactivity is shown in all three panels. Panel B: Image within the ROI in panel A reconstructed from 4 tile images of the HCN4 + /CX43 − meshwork (red) intertwined with HCN4 − /(CX43) + network (green) taken with 10x water immersion objective. Panels C: Confocal images from the area within the ROI in panel B showing: HCN4 + cells that do not express CX43 (upper image); intertwining areas between HCN4 + /(Cx43) − meshwork (red color), and penetrating HCN4-/ CX43 + cells outlined by green dots corresponding to CX43 protein on the cell membranes (middle and lower panel). Note that HCN4 expressing cells in all three images do not express CX43.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques: Immunolabeling, Expressing

    Panel A and B: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 36 tiled confocal images of the area of the 1350μm by 1350μm demonstrate meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. Gray color tissue was imaged in transmitted light. Panel B: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 16 tiled confocal images of the area of the 900μm by 900μm of another area of meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. CX43 is detected only in F-actin labeled cells.

    Journal: JACC. Clinical electrophysiology

    Article Title: Synchronized cardiac impulses emerge from heterogeneous local calcium signals within and among cells of pacemaker tissue

    doi: 10.1016/j.jacep.2020.06.022

    Figure Lengend Snippet: Panel A and B: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 36 tiled confocal images of the area of the 1350μm by 1350μm demonstrate meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. Gray color tissue was imaged in transmitted light. Panel B: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 16 tiled confocal images of the area of the 900μm by 900μm of another area of meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. CX43 is detected only in F-actin labeled cells.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques: Immunolabeling, Labeling

    Upper panels - HCN4 immunoreactive SAN cells: elongated (magenta arrows in Panel A), novel, pyramidal-like shape cells (yellow arrow in Panels B and D), spider-like (Panel C), and spindle cells (Panel E, blue arrow). Lower panels - SAN cells loaded with Fluo-4 AM have similar shapes to immunolabelled HCN4 + cells in the upper panel. Spider-like cells (Panel F) are indicated by the red arrow. Novel cells with a pyramidal-like soma (Panel G) are indicated by yellow arrows. Spindle cells are indicated by the blue arrow (Panel G and H). Elongated cells (Panel H) are indicated by magenta arrows.

    Journal: JACC. Clinical electrophysiology

    Article Title: Synchronized cardiac impulses emerge from heterogeneous local calcium signals within and among cells of pacemaker tissue

    doi: 10.1016/j.jacep.2020.06.022

    Figure Lengend Snippet: Upper panels - HCN4 immunoreactive SAN cells: elongated (magenta arrows in Panel A), novel, pyramidal-like shape cells (yellow arrow in Panels B and D), spider-like (Panel C), and spindle cells (Panel E, blue arrow). Lower panels - SAN cells loaded with Fluo-4 AM have similar shapes to immunolabelled HCN4 + cells in the upper panel. Spider-like cells (Panel F) are indicated by the red arrow. Novel cells with a pyramidal-like soma (Panel G) are indicated by yellow arrows. Spindle cells are indicated by the blue arrow (Panel G and H). Elongated cells (Panel H) are indicated by magenta arrows.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques:

    Panel A: An immunolabelled, whole mount image of a SAN preparation at low (2.5x) optical magnification. Panel B: Stacked confocal images and reconstructed front view of optically sliced z-stack images of HCN4 + /F-actin − cells (red) and HCN4 − /F-actin + cells (cyan) at a depth of 30 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective within ROI (yellow) shown in panel A. Panel C: Side views of the z-stack images in panel B, illustrating the intertwining cells of HCN4- meshwork and F-actin networks across the 30 μm depth.

    Journal: JACC. Clinical electrophysiology

    Article Title: Synchronized cardiac impulses emerge from heterogeneous local calcium signals within and among cells of pacemaker tissue

    doi: 10.1016/j.jacep.2020.06.022

    Figure Lengend Snippet: Panel A: An immunolabelled, whole mount image of a SAN preparation at low (2.5x) optical magnification. Panel B: Stacked confocal images and reconstructed front view of optically sliced z-stack images of HCN4 + /F-actin − cells (red) and HCN4 − /F-actin + cells (cyan) at a depth of 30 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective within ROI (yellow) shown in panel A. Panel C: Side views of the z-stack images in panel B, illustrating the intertwining cells of HCN4- meshwork and F-actin networks across the 30 μm depth.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques:

    3-Dimensional Images of the Cholinergic and Adrenergic Neuronal Plexus Gradients Within the HCN4 + Immunoreactive Pacemaker Cell Meshwork Mean density of adrenergic and cholinergic neuronal fibers normalized to maximum measured in 7 Z-stacks from 3 SAN preparations immunolabeled to VAChT (green) and TH (cyan) plotted against the SAN tissue deepness. The x-axes indicate tissue depth starting from 0 μm at the endocardial side and ends 300 μm at the epicardial site at. (A) Sharp gradient in which density of cholinergic innervation declines from its maximum near the endocardial site to its minimum within a distance of ~50 μm. (B) Linear gradient in which the density of cholinergic innervation declines from its maximum at the endocardial side to its minimum within a distance of ~100 μm. (C to E) Three-dimensional panoramic images of the SAN with triple immunolabeling of pacemaker cells HCN4 (red) , cholinergic VAChT (green) , and adrenergic TH (cyan) neuronal plexus separated in 3 channels. In C to E , the 3-dimensional virtual slice for Image 1 was taken at 1,000 μm from the root of the SVC, Image 2 at 1,250 μm, and Image 3 at 1,500 μm. Red arrows point to the endocardial site and black arrows point to the epicardial side of the SAN. SEPT indicates the site of septum; CRT above the yellow or red broken line indicates location of the crista terminalis; and RA indicates the right auricle. F summarizes the mean density of adrenergic and cholinergic innervation together near endocardial (ENDO) and epicardial (EPI) sites within the SAN (red bars) and within the RA (blue bars) . At the 0.05 certainty level, the mean neuronal plexus density per 0.001 mm 3 (50 μm by 100 μm by 200 μm) volume has higher density at the endocardial side of the SAN than in the auricle as tested by one-way analysis of variance. Asterisk highlights compared datasets that had showed higher density of neuronal plexus. Other abbreviations as in Figures 1 and 2 .

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: 3-Dimensional Images of the Cholinergic and Adrenergic Neuronal Plexus Gradients Within the HCN4 + Immunoreactive Pacemaker Cell Meshwork Mean density of adrenergic and cholinergic neuronal fibers normalized to maximum measured in 7 Z-stacks from 3 SAN preparations immunolabeled to VAChT (green) and TH (cyan) plotted against the SAN tissue deepness. The x-axes indicate tissue depth starting from 0 μm at the endocardial side and ends 300 μm at the epicardial site at. (A) Sharp gradient in which density of cholinergic innervation declines from its maximum near the endocardial site to its minimum within a distance of ~50 μm. (B) Linear gradient in which the density of cholinergic innervation declines from its maximum at the endocardial side to its minimum within a distance of ~100 μm. (C to E) Three-dimensional panoramic images of the SAN with triple immunolabeling of pacemaker cells HCN4 (red) , cholinergic VAChT (green) , and adrenergic TH (cyan) neuronal plexus separated in 3 channels. In C to E , the 3-dimensional virtual slice for Image 1 was taken at 1,000 μm from the root of the SVC, Image 2 at 1,250 μm, and Image 3 at 1,500 μm. Red arrows point to the endocardial site and black arrows point to the epicardial side of the SAN. SEPT indicates the site of septum; CRT above the yellow or red broken line indicates location of the crista terminalis; and RA indicates the right auricle. F summarizes the mean density of adrenergic and cholinergic innervation together near endocardial (ENDO) and epicardial (EPI) sites within the SAN (red bars) and within the RA (blue bars) . At the 0.05 certainty level, the mean neuronal plexus density per 0.001 mm 3 (50 μm by 100 μm by 200 μm) volume has higher density at the endocardial side of the SAN than in the auricle as tested by one-way analysis of variance. Asterisk highlights compared datasets that had showed higher density of neuronal plexus. Other abbreviations as in Figures 1 and 2 .

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques: Immunolabeling

    Fibrous “Cotton” Type of Anatomical Interaction Between S100B + Interstitial Cells and HCN4 + Pacemaker Cells (A) Three-dimensional image (15 μm deep) illustrates 2 unipolar S100B + cells (cyan) projecting tapered spicula that bifurcated on the HCN4-immunoreactive cells (red) . These spicula adhered so close to the HCN4 + cell that extracellular space could not be detected by optical confocal microscopy. 4’,6-Diamidino-2-phenylindole staining highlights nuclei (blue) . The soma of the unipolar S100B + cell and the bifurcations of their spicula are indicated by yellow arrows . (B) Two-dimensional image that illustrates unipolar S100B + cell, indicated by yellow arrow , connected to several HCN4 + pacemaker cells by one bifurcating spiculum. Groups of cells (yellow star) or clusters of S100B + somata (2 yellow stars) attached to HCN4 + cells and were interconnected by short extensions in a “nodal”-like net cytoarchitecture. S100B + cells from this “nodal”-like net extended long spicula to adjacent HCN4 + cells. (C) Two-dimensional image that illustrates the spiculum of an S100B + cell (cyan) that dilated in an “endfoot”-like structure, indicated by an arrow, adhering to HCN4 + pacemaker cells (red) . The 2 pacemaker cells interconnected by one bipolar S100B + interstitial cell also have a point of direct contact. (D) Three-dimensional image, 20 μm thick, that illustrates composite fibrous “cotton” connections that include the spicula, “endfeet,” and “nodal”-like net of S100B + (cyan) cells within the meshwork of HCN4 + cells (red) . Abbreviations as in Figures 2 and 4 .

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: Fibrous “Cotton” Type of Anatomical Interaction Between S100B + Interstitial Cells and HCN4 + Pacemaker Cells (A) Three-dimensional image (15 μm deep) illustrates 2 unipolar S100B + cells (cyan) projecting tapered spicula that bifurcated on the HCN4-immunoreactive cells (red) . These spicula adhered so close to the HCN4 + cell that extracellular space could not be detected by optical confocal microscopy. 4’,6-Diamidino-2-phenylindole staining highlights nuclei (blue) . The soma of the unipolar S100B + cell and the bifurcations of their spicula are indicated by yellow arrows . (B) Two-dimensional image that illustrates unipolar S100B + cell, indicated by yellow arrow , connected to several HCN4 + pacemaker cells by one bifurcating spiculum. Groups of cells (yellow star) or clusters of S100B + somata (2 yellow stars) attached to HCN4 + cells and were interconnected by short extensions in a “nodal”-like net cytoarchitecture. S100B + cells from this “nodal”-like net extended long spicula to adjacent HCN4 + cells. (C) Two-dimensional image that illustrates the spiculum of an S100B + cell (cyan) that dilated in an “endfoot”-like structure, indicated by an arrow, adhering to HCN4 + pacemaker cells (red) . The 2 pacemaker cells interconnected by one bipolar S100B + interstitial cell also have a point of direct contact. (D) Three-dimensional image, 20 μm thick, that illustrates composite fibrous “cotton” connections that include the spicula, “endfeet,” and “nodal”-like net of S100B + (cyan) cells within the meshwork of HCN4 + cells (red) . Abbreviations as in Figures 2 and 4 .

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques: Confocal Microscopy, Staining

    Anatomical Interaction Between Amoeboid-Like S100B + Interstitial Cells and HCN4 + Pacemaker Cells (A to C) Amoeboid S100B + interstitial cells (cyan) with flattened cellular extensions. Flattened S100B + extensions, or pseudopodia, were 1 to 2 μm wide and manifested dilations. S100B + pseudopodia could fold, changing the initial direction of their projection, or bifurcate and produce branches as indicated by yellow asterisks. The “plier”-like terminal dilation of an S100B + pseudopodium, enclosing an appendage from an HCN4 + pacemaker cell (red) , is indicated on A by a yellow arrow . The “patch”-like dilation of an S100B + pseudopodium that encircled a “patch” of the membrane of an HCN4 + cell is indicated by a white arrow . (D) S100B + immunoreactive cells enwrapping a group of HCN4 + pacemaker cells with a wide “ribbon”-like pseudopodium. Abbreviations as in Figures 2 and 4 .

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: Anatomical Interaction Between Amoeboid-Like S100B + Interstitial Cells and HCN4 + Pacemaker Cells (A to C) Amoeboid S100B + interstitial cells (cyan) with flattened cellular extensions. Flattened S100B + extensions, or pseudopodia, were 1 to 2 μm wide and manifested dilations. S100B + pseudopodia could fold, changing the initial direction of their projection, or bifurcate and produce branches as indicated by yellow asterisks. The “plier”-like terminal dilation of an S100B + pseudopodium, enclosing an appendage from an HCN4 + pacemaker cell (red) , is indicated on A by a yellow arrow . The “patch”-like dilation of an S100B + pseudopodium that encircled a “patch” of the membrane of an HCN4 + cell is indicated by a white arrow . (D) S100B + immunoreactive cells enwrapping a group of HCN4 + pacemaker cells with a wide “ribbon”-like pseudopodium. Abbreviations as in Figures 2 and 4 .

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques:

    3-Dimensional Images of the Cholinergic and Adrenergic Neuronal Plexus Gradients Within the HCN4 + Immunoreactive Pacemaker Cell Meshwork (A) Two-dimensional central image of hyperpolarization-activated cyclic nucleotide-gated channel 4–positive (HCN4 + ) (red) pacemaker cells and neuronal plexus. The 2 side images represent reconstructed virtual cuts of the SAN tissue from the endocardial to the epicardial side. The x-axis (pink broken line) and the y-axis (yellow broken line) on the 2-dimensional central image show the lines where the virtual cuts in the pink and yellow frames were taken. Vesicular acetylcholine transporter (VAChT) (green) and tyrosine hydroxylase (TH) (cyan) immunoreactive varicosities are co-localized with HCN4 + immunoreactive (red) pacemaker cells. (B to E) Three-dimensional images reconstructed from the series of 2-dimensional images, an example of which is shown in the central image of A. The endocardial side is on the top of the images of C to E. B displays a 3-dimensional HCN4 + meshwork (red) of the pacemaker cells seen from the endocardial side and the neuronal plexus of VAChT (green) and TH (cyan) immunoreactive neuronal fibers. In the center of the 3-dimensional image, the neuronal plexus has higher innervation than in the lower right and upper left corners. C illustrates a 3-dimensional image of the endo-epicardial gradient of cholinergic nerves. D illustrates the adrenergic innervation from the endocardial to the epicardial side. E image illustrates overlapping of the adrenergic and cholinergic innervation from the endocardial to the epicardial side. (F and G) Illustration of the meshwork of HCN4 + pacemaker cells (red) from 2 different whole-mount SAN preparations, and their associated adrenergic and cholinergic neuronal plexuses. F1 and G1 show the HCN4 + meshwork (red), F2 and G2 show the HCN4 + meshwork of pacemaker cells (red) and TH + neuronal plexus (cyan), F3 and G3 show the HCN4 + meshwork of pacemaker cells (red) and VAChT + neuronal plexus (green) , and F4 and G4 show the HCN4 + meshwork (red) , TH + neuronal plexus (cyan) , and VAChT + neuronal plexus (green) .

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: 3-Dimensional Images of the Cholinergic and Adrenergic Neuronal Plexus Gradients Within the HCN4 + Immunoreactive Pacemaker Cell Meshwork (A) Two-dimensional central image of hyperpolarization-activated cyclic nucleotide-gated channel 4–positive (HCN4 + ) (red) pacemaker cells and neuronal plexus. The 2 side images represent reconstructed virtual cuts of the SAN tissue from the endocardial to the epicardial side. The x-axis (pink broken line) and the y-axis (yellow broken line) on the 2-dimensional central image show the lines where the virtual cuts in the pink and yellow frames were taken. Vesicular acetylcholine transporter (VAChT) (green) and tyrosine hydroxylase (TH) (cyan) immunoreactive varicosities are co-localized with HCN4 + immunoreactive (red) pacemaker cells. (B to E) Three-dimensional images reconstructed from the series of 2-dimensional images, an example of which is shown in the central image of A. The endocardial side is on the top of the images of C to E. B displays a 3-dimensional HCN4 + meshwork (red) of the pacemaker cells seen from the endocardial side and the neuronal plexus of VAChT (green) and TH (cyan) immunoreactive neuronal fibers. In the center of the 3-dimensional image, the neuronal plexus has higher innervation than in the lower right and upper left corners. C illustrates a 3-dimensional image of the endo-epicardial gradient of cholinergic nerves. D illustrates the adrenergic innervation from the endocardial to the epicardial side. E image illustrates overlapping of the adrenergic and cholinergic innervation from the endocardial to the epicardial side. (F and G) Illustration of the meshwork of HCN4 + pacemaker cells (red) from 2 different whole-mount SAN preparations, and their associated adrenergic and cholinergic neuronal plexuses. F1 and G1 show the HCN4 + meshwork (red), F2 and G2 show the HCN4 + meshwork of pacemaker cells (red) and TH + neuronal plexus (cyan), F3 and G3 show the HCN4 + meshwork of pacemaker cells (red) and VAChT + neuronal plexus (green) , and F4 and G4 show the HCN4 + meshwork (red) , TH + neuronal plexus (cyan) , and VAChT + neuronal plexus (green) .

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques:

    3-Dimensional Image of the Whole-Mount SAN Preparation Showing S100B + /GFAP − Cells Three-dimensional reconstruction of the SAN from the SVC (right) to the IVC (left) and from the septum (SPT) (bottom) to the right auricle (RA) (top) 4.5 mm long, 3.5 mm wide, and 250 μm deep. Novel S100B + (cyan) /GFAP − (green) cells were detected within the HCN4 + meshwork (red) . The RA lacks S100B + (cyan) /GFAP − (green) interstitial cells. Dotted line indicates the border of crista terminalis (CT). Abbreviations as in Figures 1 and 4 .

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: 3-Dimensional Image of the Whole-Mount SAN Preparation Showing S100B + /GFAP − Cells Three-dimensional reconstruction of the SAN from the SVC (right) to the IVC (left) and from the septum (SPT) (bottom) to the right auricle (RA) (top) 4.5 mm long, 3.5 mm wide, and 250 μm deep. Novel S100B + (cyan) /GFAP − (green) cells were detected within the HCN4 + meshwork (red) . The RA lacks S100B + (cyan) /GFAP − (green) interstitial cells. Dotted line indicates the border of crista terminalis (CT). Abbreviations as in Figures 1 and 4 .

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques: Single-particle Tracking

    2-Dimensional Images of the Whole-Mount SAN Preparations With Triple Immunolabeling Illustrating PGCs and SAN Pacemaker Cells Imaged by Optical Slicing (A) Tiled panoramic 2-dimensional image (4 mm by 1.2 mm), illustrating the cytoarchitecture of the HCN4-immunoreactive meshwork from the SVC to the IVC; the approximate border of crista terminalis (CT) is indicated by the yellow broken line . Glial fibrillary acidic protein–positive (GFAP + ) (green) and S100 calcium-binding protein B–positive (S100B + ) (cyan) cells are scattered between HCN4-immunoreactive cells (red color) across the SAN. (B to E) Peripheral glial cells (PGCs) immunoreactive to GFAP and to S100B among HCN4 + cells, imaged with high optical magnification. GFAP + was detected in higher levels than S100B + within the branch PGCs. (E) Web of PGCs near the lumen of the blood vessels. Abbreviations as in Figures 1 and 2 .

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: 2-Dimensional Images of the Whole-Mount SAN Preparations With Triple Immunolabeling Illustrating PGCs and SAN Pacemaker Cells Imaged by Optical Slicing (A) Tiled panoramic 2-dimensional image (4 mm by 1.2 mm), illustrating the cytoarchitecture of the HCN4-immunoreactive meshwork from the SVC to the IVC; the approximate border of crista terminalis (CT) is indicated by the yellow broken line . Glial fibrillary acidic protein–positive (GFAP + ) (green) and S100 calcium-binding protein B–positive (S100B + ) (cyan) cells are scattered between HCN4-immunoreactive cells (red color) across the SAN. (B to E) Peripheral glial cells (PGCs) immunoreactive to GFAP and to S100B among HCN4 + cells, imaged with high optical magnification. GFAP + was detected in higher levels than S100B + within the branch PGCs. (E) Web of PGCs near the lumen of the blood vessels. Abbreviations as in Figures 1 and 2 .

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques: Immunolabeling, Binding Assay

    Variability in the Number of Detected S100B + Interstitial Cells in the Head, Body, and Tail of the SAN (A) Area near the root of the SVC, known as a “head,” of the HCN4 + meshwork of pacemaker cells (red) , from 3 different SAN preparations. (B) S100B cell populations in the “body” of the SAN between the SVC and the IVC. (C) Area close to the IVC known as the “tail” of the SAN. In all 3 panels, the upper images illustrate examples of SANs in which > 110 S100B + cells were identified, the middle images illustrate meshworks exhibiting ~60 S100B + cells, and the lower images show SANs with

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: Variability in the Number of Detected S100B + Interstitial Cells in the Head, Body, and Tail of the SAN (A) Area near the root of the SVC, known as a “head,” of the HCN4 + meshwork of pacemaker cells (red) , from 3 different SAN preparations. (B) S100B cell populations in the “body” of the SAN between the SVC and the IVC. (C) Area close to the IVC known as the “tail” of the SAN. In all 3 panels, the upper images illustrate examples of SANs in which > 110 S100B + cells were identified, the middle images illustrate meshworks exhibiting ~60 S100B + cells, and the lower images show SANs with

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques:

    2-Dimensional Images of a Whole-Mount Preparation of SAN Tissue With Triple Immunolabeling for S100B, HCN4, and TH or VAChT (A to C) Two-dimensional images of SAN tissue with triple immunolabeling for S100B + cells (cyan) , HCN4-immunoreactive pacemaker cells (red) , and TH + adrenergic fibers (green) illustrate anatomical interactions between pacemaker cells, adrenergic nerves, and interstitial cells. (D to F) Two-dimensional images of SAN tissue with triple immunolabeling for S100B + cells (cyan) , HCN4 immunoreactive pacemaker cells (red) , and VAChT + cholinergic fibers (green) illustrate anatomical relations between pacemaker cells, adrenergic nerves, and interstitial cells. Pink stars in any panel indicate the nuclei of peripheral glial cells. S100B + spicula extended from “octopus”-like cells in A, C, and E ended on TH + varicosities (A and C) or on VAChT + varicosities (E) . Yellow arrows indicate the S100B + “endfeet.” An adrenergic neurite on A , indicated by a white star , overlaps with the spiculum of an “octopus”-like S100Bþ cell. An amoeboid-like S100B + cell in the upper right corner of C receives adrenergic innervation. Images on C (adrenergic nerves) and D (cholinergic nerves) illustrate uneven innervation of S100B + cells. (F) Composite point of contact between 3 cells: one S100B-immunoreactive cell, an HCN4-immunoreactive cell, and fibers from the neuronal plexus. A white arrow indicates the region where cellular extensions from an intertwined couple of S100B + cells, an HCN4 + pacemaker cell, and a cholinergic neurite co-localize within 1 μm of each other. A white star indicates the point of contact between an S100B + cell and a cholinergic nerve, whereas a white triangle marks the point of contact between a cholinergic nerve and an HCN4 + pacemaker cell. Abbreviations as in Figures 1 , 3 , and 4 .

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: 2-Dimensional Images of a Whole-Mount Preparation of SAN Tissue With Triple Immunolabeling for S100B, HCN4, and TH or VAChT (A to C) Two-dimensional images of SAN tissue with triple immunolabeling for S100B + cells (cyan) , HCN4-immunoreactive pacemaker cells (red) , and TH + adrenergic fibers (green) illustrate anatomical interactions between pacemaker cells, adrenergic nerves, and interstitial cells. (D to F) Two-dimensional images of SAN tissue with triple immunolabeling for S100B + cells (cyan) , HCN4 immunoreactive pacemaker cells (red) , and VAChT + cholinergic fibers (green) illustrate anatomical relations between pacemaker cells, adrenergic nerves, and interstitial cells. Pink stars in any panel indicate the nuclei of peripheral glial cells. S100B + spicula extended from “octopus”-like cells in A, C, and E ended on TH + varicosities (A and C) or on VAChT + varicosities (E) . Yellow arrows indicate the S100B + “endfeet.” An adrenergic neurite on A , indicated by a white star , overlaps with the spiculum of an “octopus”-like S100Bþ cell. An amoeboid-like S100B + cell in the upper right corner of C receives adrenergic innervation. Images on C (adrenergic nerves) and D (cholinergic nerves) illustrate uneven innervation of S100B + cells. (F) Composite point of contact between 3 cells: one S100B-immunoreactive cell, an HCN4-immunoreactive cell, and fibers from the neuronal plexus. A white arrow indicates the region where cellular extensions from an intertwined couple of S100B + cells, an HCN4 + pacemaker cell, and a cholinergic neurite co-localize within 1 μm of each other. A white star indicates the point of contact between an S100B + cell and a cholinergic nerve, whereas a white triangle marks the point of contact between a cholinergic nerve and an HCN4 + pacemaker cell. Abbreviations as in Figures 1 , 3 , and 4 .

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques: Immunolabeling

    2-Dimensional Images of S100B + /GFAP − Interstitial Cells and PGCs Embedded Within the HCN4 + Meshwork Pink arrows indicate S100B + /GFAP − interstitial cells, and yellow arrows indicate S100B + /GFAP + PGCs. (A to D) S100B + /GFAP − interstitial cells (cyan) between, and in close proximity to, HCN4 + pacemaker cells (red) . Higher levels of GFAP (green) than S100B were detected in the extensions of PGCs. (E) A 2-dimensional image of the radiating branches of adrenergic TH + (green) fibers as well as S100B + interstitial cells (cyan) , among HCN4-immunoreactive pacemaker cells (red) . (F) A 2-dimensional image of the radiating branches of cholinergic VAChT + (green) fibers and S100B + interstitial cells (cyan) , among HCN4-immunoreactive pacemaker cells (red) . Abbreviations as in Figures 2 and 4 .

    Journal: JACC. Clinical electrophysiology

    Article Title: The Heart’s Pacemaker Mimics Brain Cytoarchitecture and Function

    doi: 10.1016/j.jacep.2022.07.003

    Figure Lengend Snippet: 2-Dimensional Images of S100B + /GFAP − Interstitial Cells and PGCs Embedded Within the HCN4 + Meshwork Pink arrows indicate S100B + /GFAP − interstitial cells, and yellow arrows indicate S100B + /GFAP + PGCs. (A to D) S100B + /GFAP − interstitial cells (cyan) between, and in close proximity to, HCN4 + pacemaker cells (red) . Higher levels of GFAP (green) than S100B were detected in the extensions of PGCs. (E) A 2-dimensional image of the radiating branches of adrenergic TH + (green) fibers as well as S100B + interstitial cells (cyan) , among HCN4-immunoreactive pacemaker cells (red) . (F) A 2-dimensional image of the radiating branches of cholinergic VAChT + (green) fibers and S100B + interstitial cells (cyan) , among HCN4-immunoreactive pacemaker cells (red) . Abbreviations as in Figures 2 and 4 .

    Article Snippet: HCN4+ cells were identified by rabbit polyclonal antibodies for cyclic nucleotide-gate cation channels HCN4 (1:300; Alomone Labs).

    Techniques:

    Single molecule mass photometry of HCN4—EGFP-TRIP8b complex. (A) SEC of HCN4—EGFP-TRIP8b complex following purification in LMNG/CHS. Peak fractions used for MP are delimited by the red lines. (B) SDS-PAGE gel of the pooled SEC fractions of HCN4—EGFP-TRIP8b complex, stained with Coomassie blue. Filled arrows indicate HCN4, while open arrows indicate EGFP-TRIP8b, in their oligomeric (square bracket), and monomeric forms. (C) Western blots of the pooled SEC fractions of HCN4—EGFP-TRIP8b complex performed by using anti-TRIP8b and anti-HCN4 antibodies, respectively. Open and filled arrows as in panel (B) . Of note that the western blot anti-TRIP8b reveals the presence of a small number of molecules (faint band between 60 and 50 kDa markers) corresponding N terminally degraded TRIP8b. Indeed, they co-purified with HCN4 and thus have retained the HCN binding sites located in their C-terminal half ( Santoro et al., 2004 ; Santoro et al., 2011 ). (D) Mass histogram of binding events for HCN4—EGFP-TRIP8b complex before (left) and after the addition of 2 mM cAMP (right). The blue lines represent the fitting of the data with Gaussian Distribution Function. Left, schematic representation of purified HCN4 (green) embedded into a detergent micelle (yellow) and bound to four TRIP8b molecules (blue). Right, purified HCN4 bound to cAMP (red) has lost TRIP8b.

    Journal: Frontiers in Physiology

    Article Title: Validation of the binding stoichiometry between HCN channels and their neuronal regulator TRIP8b by single molecule measurements

    doi: 10.3389/fphys.2022.998176

    Figure Lengend Snippet: Single molecule mass photometry of HCN4—EGFP-TRIP8b complex. (A) SEC of HCN4—EGFP-TRIP8b complex following purification in LMNG/CHS. Peak fractions used for MP are delimited by the red lines. (B) SDS-PAGE gel of the pooled SEC fractions of HCN4—EGFP-TRIP8b complex, stained with Coomassie blue. Filled arrows indicate HCN4, while open arrows indicate EGFP-TRIP8b, in their oligomeric (square bracket), and monomeric forms. (C) Western blots of the pooled SEC fractions of HCN4—EGFP-TRIP8b complex performed by using anti-TRIP8b and anti-HCN4 antibodies, respectively. Open and filled arrows as in panel (B) . Of note that the western blot anti-TRIP8b reveals the presence of a small number of molecules (faint band between 60 and 50 kDa markers) corresponding N terminally degraded TRIP8b. Indeed, they co-purified with HCN4 and thus have retained the HCN binding sites located in their C-terminal half ( Santoro et al., 2004 ; Santoro et al., 2011 ). (D) Mass histogram of binding events for HCN4—EGFP-TRIP8b complex before (left) and after the addition of 2 mM cAMP (right). The blue lines represent the fitting of the data with Gaussian Distribution Function. Left, schematic representation of purified HCN4 (green) embedded into a detergent micelle (yellow) and bound to four TRIP8b molecules (blue). Right, purified HCN4 bound to cAMP (red) has lost TRIP8b.

    Article Snippet: Primary antibody dilutions were as follows: anti-HCN4 (rabbit polyclonal, Alomone) 1:1000; anti-TRIP8b (mouse monoclonal, NeuroMab) 1:1000.

    Techniques: Purification, SDS Page, Staining, Western Blot, Binding Assay

    Functional characterization of the inhibitory effect of TRIP8b on the HCN4 construct employed for MP. (A) Representative whole-cell current traces of HCN4 channels recorded, with 0.25 µM cAMP in the patch pipette, in HEK293T cells transiently expressing the channel alone (top) or with GFP-TRIP8b (1a) (bottom). Black arrows indicate the current selected for analysis in (B) . (B) Mean activation curves of HCN4 channels alone (black full circles) or co-expressed with GFP-TRIP8b (1a) (black open circles) with cAMP in the patch pipette obtained from tail currents collected at −40 mV (see arrows in panel (A) . Dashed lines indicate Boltzmann fitting to the data (see Materials and methods) from which the half activation potential (V 1/2 ) were derived. HCN4 + cAMP = −76.9 ± 0.6 mV; HCN4 + TRIP8b + cAMP = −94.2 ± 1.2 mV. Data are presented as mean ± SEM. Number of cells (N) ≥ 8. The two half activation potentials are statistically different. Statistical analysis performed with t-student test ( p

    Journal: Frontiers in Physiology

    Article Title: Validation of the binding stoichiometry between HCN channels and their neuronal regulator TRIP8b by single molecule measurements

    doi: 10.3389/fphys.2022.998176

    Figure Lengend Snippet: Functional characterization of the inhibitory effect of TRIP8b on the HCN4 construct employed for MP. (A) Representative whole-cell current traces of HCN4 channels recorded, with 0.25 µM cAMP in the patch pipette, in HEK293T cells transiently expressing the channel alone (top) or with GFP-TRIP8b (1a) (bottom). Black arrows indicate the current selected for analysis in (B) . (B) Mean activation curves of HCN4 channels alone (black full circles) or co-expressed with GFP-TRIP8b (1a) (black open circles) with cAMP in the patch pipette obtained from tail currents collected at −40 mV (see arrows in panel (A) . Dashed lines indicate Boltzmann fitting to the data (see Materials and methods) from which the half activation potential (V 1/2 ) were derived. HCN4 + cAMP = −76.9 ± 0.6 mV; HCN4 + TRIP8b + cAMP = −94.2 ± 1.2 mV. Data are presented as mean ± SEM. Number of cells (N) ≥ 8. The two half activation potentials are statistically different. Statistical analysis performed with t-student test ( p

    Article Snippet: Primary antibody dilutions were as follows: anti-HCN4 (rabbit polyclonal, Alomone) 1:1000; anti-TRIP8b (mouse monoclonal, NeuroMab) 1:1000.

    Techniques: Functional Assay, Construct, Transferring, Expressing, Activation Assay, Derivative Assay

    Alteration in HCN channel expression in thalamus following general demyelination. ( A , B ) Immunofluorescence staining of the VB complex (horizontal thalamic sections, 40 μm) comparing the expression of HCN4 (A, in red, rb-anti-HCN4, 1:200, Alomone) and HCN2 (B, in red, rb-anti-HCN4, 1:200, Alomone) channels between control C3H/HeJ and Day1. The purified ms-anti- neurofilament antibody (SMI312, pan axonal, 1:200, BioLegend) was used as an axonal marker (SMI312, in green). Cell nuclei were stained with DAPI (in blue). ( C , D ) Bar graphs comparing the intensity of the fluorescence signal (using integrated fluorescence intensity values) for SMI312 (C and D upper traces) and HCN4 and HCN2 (lower traces) between the control C3H/HeJ and Day1. ( E ) Immunofluorescence staining of VB in control C3H/HeJ and Day1 with antibodies against TRIP8b (ms-anti-(constant) TRIP8b, 1:50, NeuroMab, in green) and phosphorylated TRIP8b (rb-α-pS237 antibody, 1:100, YenZym). ( F ) Representative bar graph comparing the intensity of the fluorescence signal for total TRIP8b and pS237 between the two groups indicating a significant reduction for phosphorylated TRIP8b, pS237, on Day1. Scale bars indicate 100 μm. VPL, VPM, and TRN stand for ventral-posterior medial, ventral-posterior lateral, and thalamic reticular nucleus, respectively.

    Journal: Cerebral Cortex (New York, NY)

    Article Title: Modulation of pacemaker channel function in a model of thalamocortical hyperexcitability by demyelination and cytokines

    doi: 10.1093/cercor/bhab491

    Figure Lengend Snippet: Alteration in HCN channel expression in thalamus following general demyelination. ( A , B ) Immunofluorescence staining of the VB complex (horizontal thalamic sections, 40 μm) comparing the expression of HCN4 (A, in red, rb-anti-HCN4, 1:200, Alomone) and HCN2 (B, in red, rb-anti-HCN4, 1:200, Alomone) channels between control C3H/HeJ and Day1. The purified ms-anti- neurofilament antibody (SMI312, pan axonal, 1:200, BioLegend) was used as an axonal marker (SMI312, in green). Cell nuclei were stained with DAPI (in blue). ( C , D ) Bar graphs comparing the intensity of the fluorescence signal (using integrated fluorescence intensity values) for SMI312 (C and D upper traces) and HCN4 and HCN2 (lower traces) between the control C3H/HeJ and Day1. ( E ) Immunofluorescence staining of VB in control C3H/HeJ and Day1 with antibodies against TRIP8b (ms-anti-(constant) TRIP8b, 1:50, NeuroMab, in green) and phosphorylated TRIP8b (rb-α-pS237 antibody, 1:100, YenZym). ( F ) Representative bar graph comparing the intensity of the fluorescence signal for total TRIP8b and pS237 between the two groups indicating a significant reduction for phosphorylated TRIP8b, pS237, on Day1. Scale bars indicate 100 μm. VPL, VPM, and TRN stand for ventral-posterior medial, ventral-posterior lateral, and thalamic reticular nucleus, respectively.

    Article Snippet: Sections were washed three times for 10 min in PBS and incubated for 2 h in blocking solution (10% normal goat serum, 3% bovine serum albumin (BSA), 0.3% Triton-X100 in PBS) followed by 48 h of incubation at 4 °C with the following primary antibodies: polyclonal rabbit (rb)-anti-HCN2 (1:200), rb-anti-HCN4 (1:200; Alomone Labs), rb-anti-NeuN (neuronal specific marker, 1:1000; Abcam), mouse purified (ms)-anti-neurofilament marker (pan axonal, cocktail, SMI312; 1:200, BioLegend) and ms-anti-Parvalbumin (PV235, 1:500; Swant).

    Techniques: Expressing, Immunofluorescence, Staining, Purification, Marker, Fluorescence