DOI: 10.5061/dryad.k98sf7mmp
title: Data from: Microvascular rarefaction in the sinoatrial node

description: This study tested the hypothesis that sinoatrial node microvascular
 rarefaction occurs in a model of heart failure with preserved ejection
 fraction (HFpEF). While it has been established that the
 sinoatrial node exhibits vascular heterogeneity, it was not known whether
 microvessel density changes regionally or is altered overall during HFpEF.
 Data provided here demonstrates vascular and pacemaker cell densities
 under normal conditions with representations at low and high
 magnifications. 
description: <strong>Whole-Mount
 Immunohistochemistry</strong> Following
 confirmation of isoflurane anesthesia, we rapidly excised hearts and
 placed them in prewarmed (37 °C) heparinized (0.2 mg/mL) Tyrode III
 solution (in mM: 140 NaCl, 5.4 KCl, 1.0 MgCl₂, 1.8 CaCl₂, 5.0 HEPES, and
 10 d-glucose, pH adjusted to 7.4 with NaOH). We then dissected the SAN and
 surrounding regions, including portions of the right atrial appendage,
 superior vena cava, and inferior vena cava. We pinned and stretched the
 tissues in a 35 mm Sylgard-coated dish, rapidly rinsed them three times in
 phosphate-buffered saline (PBS), and fixed them in 2 mL of 4%
 paraformaldehyde (0.01 M PBS) for 1 hour at room temperature. After
 fixation, we washed the tissues three times with PBS and transferred them
 to a 50 mL tube containing PBS for 12 hours at 4°C. We performed tissue
 dehydration, bleaching, and clearing in dimethyl sulfoxide (DMSO) to
 reduce tissue autofluorescence, as previously described [PMID: 25262932,
 16806289]. Briefly, we dehydrated tissues through an EtOH gradient (25,
 50, 75, 95, 100%), cleared for 2 hours in 20% DMSO in EtOH, and bleached
 tissues for 12 hours in 6% hydrogen peroxide in EtOH. We achieved
 rehydration via a reverse ethanol gradient, followed by three 10-minute
 PBS washes. We permeabilized tissues with 0.5% Triton X-100 in PBS (3 x
 10min). We blocked non-specific binding with 5% normal donkey serum in
 0.5% Triton X-100 PBS for 2 hours. After PBS washes (3 x 10min), we
 incubated the tissues for 48 hours at 4 °C in a 200μL volume of primary
 antibody cocktail, which included: goat anti-mouse CD31 (1:50; AF3628,
 R&D Systems) and rabbit anti-mouse HCN4 (1:200; ab289962, Abcam)
 diluted in PBS. We washed primary antibodies three times in PBS followed
 by incubation in 1 mL of secondary antibodies cocktail containing donkey
 anti-goat Alexa Fluor 488 (1:1000, ab150129, Abcam), and donkey
 anti-rabbit Alexa Fluor 594, (1:1000, ab150076; Abcam) for 3 hours at room
 temperature with minimal light exposure. After labeling, we washed tissues
 in PBS, and incubated them for 2 hours in a 25% DMSO PBS solution. We then
 mounted tissues in Vectashield vibrance medium (Vector Laboratories) with
 a coverslip and allowed mounting media hardening for 12 hours before
 acquiring images. <strong>Immunohistochemistry
 Image Acquisition</strong> For confocal tissue
 imaging, we used an inverted Leica Stellaris 5 microscope (Leica
 Microsystems, Wetzlar, Germany) equipped with 10x objective (Leica HC PL
 APO 10x, NA 0.40 CS2) for imaging the entire dissected tissue, 20x
 objective (Leica HCX PL FL 20x, NA 0.40 CS2) for focusing on the entire
 SAN region and, 63x oil-immersion objective (Leica HC PL APO 63x, NA = 1.4
 Oil CS2) for specific sSAN and iSAN regions. We captured sSAN and iSAN
 images at 0.18 x 0.18x 0.30 μm/pixel resolution (231
 μm<sup>2</sup> field of view) using Leica Application Suite X
 (LAS X) software. We acquired Z-stacks to span the entire SAN tissue depth
 (approximately 406 Z-planes). We used the LAS X Navigator to perform
 stitching with an overlap of approximately 15%.
description: # Data from: Microvascular rarefaction in the sinoatrial node Dataset DOI:
 [10.5061/dryad.k98sf7mmp](https://doi.org/10.5061/dryad.k98sf7mmp) ##
 Description of the data and file structure This dataset contains
 immunofluorescent images of the cardiac sinoatrial node labeled with
 antibodies against HCN4 and CD31. HCN4 labels cardiac pacemaker cells, and
 CD31 labels endothelial cells. Images were taken at 10X, 20X, and 60X. For
 images taken at 10X, the entire cardiac sinoatrial node is visualized
 along with surrounding tissue, including the right atrial appendage,
 superior vena cava, and inferior vena cava. Multiple images are taken and
 stitched together. For the 20X image, just the region encompassing the
 cardiac sinoatrial node is acquired, where multiple tiled images are
 stitched together. For the 60X images, single areas of the superior (sSAN)
 or inferior (iSAN) sinoatrial nodes were acquired. ### Files and variables
 #### File: 20241210-HCN4-CD31-10X-Tile.tif **Description:** Cardiac
 sinoatrial node tissue immunofluorescently labeled with antibodies against
 HCN4 (HCN4-expressing pacemaker cells) and CD31 (endothelial cells in the
 endocardium and vascular endothelium). Image taken at 10x magnification
 and tiled to image the entire sinoatrial node. #### File:
 20241210-HCN4-CD31-20X-Tile.tif **Description:** Cardiac sinoatrial node
 tissue immunofluorescently labeled with antibodies against HCN4
 (HCN4-expressing pacemaker cells) and CD31 (endothelial cells in the
 endocardium and vascular endothelium). Image taken at 20x magnification
 and tiled to image the entire sinoatrial node. #### File:
 20241210-HCN4-CD31-iSAN-60X.tif **Description:** Inferior region of the
 cardiac sinoatrial node tissue immunofluorescently labeled with antibodies
 against HCN4 (HCN4-expressing pacemaker cells) and CD31 (endothelial cells
 in the endocardium and vascular endothelium). Image taken at 60x
 magnification of a sub-region of the node. #### File:
 20241210-HCN4-CD31-sSAN-60X.tif **Description:** Superior region of the
 cardiac sinoatrial node tissue immunofluorescently labeled with antibodies
 against HCN4 (HCN4-expressing pacemaker cells) and CD31 (endothelial cells
 in the endocardium and vascular endothelium). Image taken at 60x
 magnification of a sub-region of the node. ## Code/software Open-source
 software, such as ImageJ/FIJI, can be used to view the data. Images can be
 opened directly in ImageJ/FIJI using the Bioformats plugin.