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2 protocols using anti dna rna damage

1

Quantifying Endothelial and Cardiomyocyte Stress

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Flow cytometric analysis of endothelial and cardiomyocyte oxidative stress and IL-36R was performed as previously described (15 (link)). Briefly, hearts were collagenase-digested to obtain single-cell suspensions. Cells were incubated with antibodies: anti-DNA/RNA damage (1:100 dilution, Abcam), anti-IL-36R (1:100 dilution, R & D Systems; Alexa-647 secondary, 1:100 dilution, Biolegend), anti-CD31 (1:100 dilution, Biolegend), anti-cTnT (1:100 dilution, Miltenyi Biotec), and Zombie dye (1:500 dilution, Biolegend), along with appropriate IgG controls. Flow cytometry using a CyAn™ ADP instrument (Beckman Coulter, USA) captured 250,000 events per sample. Data analysis was performed with Summit 4.3 software (Beckman Coulter, USA).
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2

Multicolor Immunofluorescence Staining Protocol

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Immunofluorescence was performed as described previously2 (link). The following antibodies or chemical compounds were used: COL17A1 (Abcam, ab186415, 1:400), Keratin 14 (Biolegend, 906004, 1:500), ITGA6 (BD, 555734, 1:200), Keratin 15 (Biolegend, 833904, 1:300), NFκB (Santa Cruz, sc-101749, 1:50), CD11b (BD, 557686, 1:100), F4/80 (Bio-Rad, MCA497A488, 1:100), KI67 (Invitrogen, 14-5698-82, 1:300), CD3 (Biolegend, 100235, 1:100), MHC2 (Biolegend, 107605, 1:100), CD31(BD Biosciences, 557355, 1:200), anti-DNA/RNA Damage (Abcam, ab62623, 1:2000), Tom20 (Thermo Fisher, 11802-1-AP, 1:30), Keratin 1 (Abcam, ab185628, 1:300), survivin (Cell signaling, 2808, 1:100), casp3 (Cell signaling, 9661, 1:200), Tuj1 (Covance, PRB-435P, 1:500), CD34-FITC (Invitrogen, 11-0341-85, 1:100), ITGA6-PE (BD, 555736, 1:100), Sca1-APC (Miltenyi Biotec, 130-102-343, 1:100), CD45-APC-cy7 (BioLegend, 103116, 1:100). FV10-ASW4.2 was used for capturing confocal images. CellSens Standard v1.13 (Olympus) was used for collecting HE data.
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