All steps were performed at 4 °C to ensure integrity of chromatin and RNA. For nuclear RNA isolation RNAsin (80 U/ml, Promega) was added to all buffers. Isolated nuclei were stained in 500 µl staining buffer (phosphate-buffered saline (PBS) containing 1 % bovine serum albumin (BSA), 22.5 mg/ml glycine, 0.1% Tween 20) using anti-PCM1 (1:500, HPA023370, Sigma) and anti-PLN antibodies (1:500, A010–14, Badrilla) for 30 min. For isotype control stainings, we used primary antibodies lacking target specificity (1:1000, anti-mouse, 554121, BD; 1:1000, anti-rabbit, Z25308, Life technologies). Subsequently, the corresponding Alexa488- and Alexa568-labeled secondary antibodies (1:1000, A11029 and A11011, Invitrogen) were added. After 30 min of incubation, nuclei were pelleted by centrifugation (1000 × g, 5 min) and resuspended in 1 ml PBS containing 1 mM ethylenediaminetetraacetic acid (EDTA). Nuclei were filtered (CellTrics 30 µm, Sysmex) and incubated with Draq7 (final concentration 2.25 nM, Cell Signaling) for 10 min. Nuclei were analyzed (Bio-Rad S3, Bio-Rad; LSRFortessa, BD) and sorted by flow cytometry (Bio-Rad S3, Bio-Rad).
Hpa023370
Manufactured by Merck Group
Sourced in United States
HPA023370 is a laboratory equipment product manufactured by Merck Group. It is a specialized instrument designed for specific scientific applications. The core function of this product is to perform tasks that are essential for research and analysis purposes. However, a detailed and unbiased description of the product's specific capabilities and intended use cannot be provided without the risk of extrapolation.
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Lab products found in correlation
Texas red, by Thermo Fisher Scientific (4 mentions)
Collagenase type 1, by Merck Group (2 mentions)
Fluoromount g with dapi, by Southern Biotech (2 mentions)
Eclipse ti inverted microscope, by Nikon (2 mentions)
A11011, by Thermo Fisher Scientific (1 mentions)
Draq7, by Cell Signaling Technology (1 mentions)
Biorad s3, by Bio-Rad (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Rnasin, by Promega (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
A7811, by Merck Group (1 mentions)
Ki67 clone sp6, by Abcam (1 mentions)
Mouse on mouse m o m basic kit, by Vector Laboratories (1 mentions)
Cryostat, by Leica (1 mentions)
Fv1000, by Olympus (1 mentions)
Bz x710, by Keyence (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
15 protocols using hpa023370
1
Isolation and Analysis of Nuclei for RNA Studies
2
Immunostaining of Cardiac Tissue Sections
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Frozen heart sections embedded in OCT compound (Tissue-Tek; Sakura, UAE) were cut into 8 μm sections with a cryostat (Leica, Germany), permeabilized, blocked with Blocking-One (Nacalai Tesque, Japan), and labeled with primary antibodies, followed by fluorochrome-conjugated secondary antibodies. Counterstaining for DAPI (nuclei), phalloidin (F-actin), and wheat germ agglutinin (WGA; cell membrane) was also performed. Sections were covered with a fluorescence mounting medium (Dako, USA) and examined using an inverted fluorescence microscope (BZ-X710, Keyence, Japan), or a confocal scanning system mounted on a IX81 inverted microscope (FV-1000, Olympus, Japan) (Hashimoto et al., 2018 (link)). The primary antibodies used were for Ki67 (clone SP6, Abcam, UK), phospho-histone H3 at Ser-10 (06-570, EMD Millipore, USA), PCM-1 (HPA023370, Sigma-Aldrich), sarcomeric α-actinin (A7811, Sigma-Aldrich), and the FLAG tag (F1804, Sigma-Aldrich). When using mouse-derived antibodies, the Mouse on Mouse (M.O.M.) Basic Kit (Vector, CA, USA) was used. Essentially the same staining protocol was applied for CMs from aged mice isolated with a fixation digestion method (see below). In freshly isolated fetal CMs, fixation was done with 4% paraformaldehyde before permeabilization.
3
Quantifying Myocyte Cross-Sectional Area
4
Quantifying Myocyte Cross-Sectional Area
5
Quantifying Myocyte Cross-Sectional Area
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To determine myocyte cross-sectional area, thin myocardial sections were stained with wheat germ agglutinin (WGA) as published[30 (link),36 (link)]. Briefly, 5 μm thin paraffin sections were incubated with WGA (1 μg/mL) conjugated to Texas red (Thermo Fisher Scientific, Cat# W21405). To identify cardiac myocytes, sections were co-stained with an antibody against pericentriolar material protein 1 (PCM1), which specifically marks myocytes in the heart, at 1:1000 dilution in a blocking buffer overnight at 4 °C (Sigma Cat# HPA023370)[30 (link),36 (link)–38 (link)]. Sections were then stained with DAPI, mounted using a fluorescent mounting media, and imaged, as described above. WGA-stained images were analyzed using Image J software (https://imagej.net ). Total pixels obtained for WGA staining were subtracted from the total pixel count per field. The residual pixel count was divided by an average number of cardiac myocytes identified by PCM1 staining in each field. An average of 10 to 15 fields per section, 5–6 sections per heart, representing around 15,000 to 20,000 nuclei per heart. About 4–7 mice per genotype were used to calculate the mean myocyte cross-sectional area.
6
Histological Cardiac Assessment Techniques
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Hearts were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), embedded in paraffin, cut into 3 μm slices and stained with haematoxylin–eosin, Sirius-red or fluorescent wheat germ agglutinin (WGA; Alexa Fluor 488 conjugate, Invitrogen, Karlsruhe, Germany). Nuclei were counterstained with propidium iodide (Sigma, Munich, Germany). For immunohistochemical staining, hearts were snap frozen, embedded in optimum cutting temperature (OCT) medium (Tissue-Tek), cut into 3 μm slices and stained with an antibody against PCM1 (1:1,000, HPA023370, Sigma) in combination with a Cy3-labelled anti-rabbit antibody (1:1,000, Invitrogen). Glycocalyx was stained with WGA (Alexa Fluor 488 conjugate, Invitrogen) and nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole, Invitrogen).
7
Cardiomyocyte Nuclei Isolation and Analysis
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Cardiomyocyte nuclei were isolated from cardiac tissue20 (link) and were stained by an antibody directed against PCM1 (1:1,000, HPA023370, Sigma) and an Alexa488-labelled secondary anti-rabbit antibody (1:1,000, Invitrogen) or a molecular beacon targeting mRNA43 (link) of Tnnt2 (Supplementary Data 3 ; 50 nM) for 30 min. Nuclei were identified by 7-aminoactinomycin D (7-AAD) (1:500, Invitrogen). For analysis of αMHC-H2B-mCherry transgenic mice, an Alexa647-labelled secondary anti-rabbit antibody (1:1,000, Invitrogen) and for nuclei-staining DAPI (1:1,000, Invitrogen) was used. Nuclei were analysed or sorted by flow cytometry (CyFlow Space, Partec, Münster, Germany; BD Influx cell sorter, BD Biosciences, Heidelberg, Germany; Bio-Rad S3 cell sorter, Bio-Rad Laboratories, Munich, Germany).
8
Immunostaining of Myoblast Differentiation
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Proliferating cells were fixed on a coverslip at about 70% confluence, differentiating cells at the respective timepoints of interest. The cells were fixed with ice-cold methanol or 4% PFA in PBS for 15 min. Before staining, PFA fixed cells were permeabilized with 0.1% Triton X-100 for 5 min. As a myogenic marker, cells were stained for desmin (NBP1-45143, 1:50; or NSJ-R31530, 1:50). To check the proliferation status, cells were stained for Ki-67 (SAB5500134, 1:1000). To differentiate between myoblasts and fibroblasts, we also stained against fibronectin (SC-59826, 1:50). For myoblast differentiation, we stained at different timepoints for myosin heavy chain (NovocastraTM, NCL-MHCf, 1:100) and PCM1 (Sigma HPA023370, 1:100). Immunofluorescence pictures were taken with an Olympus IX83 microscope and U Plan X Apo 40× or U Plan X Apo 10× objective. The cell counter function of Image J [22 (link)] was used to determine the number of cells (DAPI), as well as the number of cells which were positive for desmin and/or Ki-67.
9
Quantifying Myocyte Cross-Sectional Area
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To quantitate myocyte cross-sectional area (CSA), thin myocardial sections were stained with wheat germ agglutinin (WGA) to mark the interstitium, and an antibody against pericentriolar material protein 1 (PCM1), which marks the myocyte nuclei (Sigma, Cat# HPA023370), as published[23 (link),24 (link),30 ,31 (link)]. In brief, thin myocardial sections (5 μm) were incubated with WGA (1 μg/mL) conjugated to Texas red (Thermo Fisher Scientific, Cat# W21405) to mark the interstitium. Sections were co-stained with an anti PCM1 antibody and DAPI. The WGA and PCM1 co-stained images were analyzed by Image J software (https://imagej.net ) to measure the interstitial area and to count the number of cardiac myocytes in each field, normalized to the nuclei stained positive for PCM1 expression. An average of 8 fields per section, 6 sections per heart, representing around 20,000 nuclei per heart was analyzed.
10
Immunohistochemistry of Cardiac Proteins
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Immunohistochemistry was performed on optimal cutting temperature compound–embedded frozen sections (Sakura 4583) with a combination of sarcomeric α-actinin antibody (1:100, Abcam ab9465), NaV1.5 antibody (1:100, Cell Signaling Technology 14421), PCM1 antibody (1:1,000, Sigma HPA023370), active β-catenin (ABC) antibody (1:50, Developmental Studies Hybridoma Bank, University of Iowa PY489-B-catenin), pan β-catenin antibody (1:100, BD 610153), and Cx43 antibody (1:50, Thermo Fisher Scientific 71-0700). Secondary antibodies included Alexa Fluor 488 (1:200, Abcam ab150077, ab150113, and ab150121) and Alexa Fluor 568 (1:200, Abcam ab175471). Slides were treated with TrueBlack Lipofuscin Autofluorescence Quencher (Biotium) and then with DAPI (Sigma 28718-90-3).
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