hcn4  (Alomone Labs)


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    Structured Review

    Alomone Labs hcn4
    Panels A and B : Optical slices (1 μm thick) of <t>HCN4</t> 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.
    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
    https://www.bioz.com/result/hcn4/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hcn4 - by Bioz Stars, 2022-11
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    Images

    1) Product Images from "Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue"

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    Journal: bioRxiv

    doi: 10.1101/2020.04.14.039461

    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.
    Figure Legend 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.

    Techniques Used: Microscopy, Immunolabeling

    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. 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.
    Figure Legend 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. 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.

    Techniques Used: Immunolabeling

    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.
    Figure Legend 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.

    Techniques Used: Immunolabeling, Labeling

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: Immunolabeling, Expressing

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    Alomone Labs cyclic nucleotide gate cation channels hcn4
    3-Dimensional Images of the Cholinergic and Adrenergic Neuronal Plexus Gradients Within the <t>HCN4</t> + 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 <t>channels.</t> 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 .
    Cyclic Nucleotide Gate Cation Channels Hcn4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs guinea pig anti hcn4 antibody
    3-Dimensional Images of the Cholinergic and Adrenergic Neuronal Plexus Gradients Within the <t>HCN4</t> + 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 <t>channels.</t> 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 .
    Guinea Pig Anti Hcn4 Antibody, 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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    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

    Effect of chronic training on HCN1, KCN2 and HCN4 protein expression determined by immunocytochemical techniques in sedentary and trained dog. Bar diagram on top left indicates that the relative density of dog cardiomyocytes with HCN4 immunolabelling obtained from the trained group (n=30 cells/6 dogs) is significantly increased compared to that measured in the sedentary group (n=30 cells/6 dogs). Original immunofluorescent images are shown on the left . Bottom panels indicate lack of effect of chronic training on the relative density of dog cardiomyocytes with HCN1 and HCN2 immunolabelling (n=30 – 30 cells/6 – 6 dogs for sedentary and trained groups, respectively). Figure 7–Source Data 1 Effect of chronic training on HCN4 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 2 Effect of chronic training on HCN1 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 3 Effect of chronic training on HCN2 protein expression determined by immunocytochemical technique in sedentary and trained dogs.

    Journal: bioRxiv

    Article Title: Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibilty in a canine model of elite exercise

    doi: 10.1101/2022.07.13.499876

    Figure Lengend Snippet: Effect of chronic training on HCN1, KCN2 and HCN4 protein expression determined by immunocytochemical techniques in sedentary and trained dog. Bar diagram on top left indicates that the relative density of dog cardiomyocytes with HCN4 immunolabelling obtained from the trained group (n=30 cells/6 dogs) is significantly increased compared to that measured in the sedentary group (n=30 cells/6 dogs). Original immunofluorescent images are shown on the left . Bottom panels indicate lack of effect of chronic training on the relative density of dog cardiomyocytes with HCN1 and HCN2 immunolabelling (n=30 – 30 cells/6 – 6 dogs for sedentary and trained groups, respectively). Figure 7–Source Data 1 Effect of chronic training on HCN4 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 2 Effect of chronic training on HCN1 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 3 Effect of chronic training on HCN2 protein expression determined by immunocytochemical technique in sedentary and trained dogs.

    Article Snippet: After the incubation period, cells were labelled overnight at 4°C with anti-KChIP2 (Alomone, #APC-142, RRID:AB_2756744), anti-Kv4.3 (Alomone, #APC-017, RRID:AB_2040178), anti-HCN1 (Alomone, #APC-056, RRID:AB_2039900), anti-HCN2 (Alomone, #APC-030, RRID:AB_2313726) and anti-HCN4 (Alomone, #APC-052, RRID:AB_2039906) primary antibody diluted to 1:50.

    Techniques: Expressing