Western blot analysis was performed on total crude protein lysates from human tissue, 2D‐cultured hiPSC‐CMs, and EHTs. Lysates of single samples or pooled samples were separated on 10% acrylamide/bisacrylamide (29:1) gels and transferred by wet electroblotting to nitrocellulose membranes. Membranes were stained with primary antibodies directed against α‐actinin 2 (1:1,000, Sigma) and cardiac troponin T (1:5,000, Abcam). Peroxidase‐conjugated secondary antibodies against mouse (1:5,000, Sigma) or against rabbit (1:6,000, Sigma) were used. Signals were revealed with the Clarity Western ECL Substrate (Bio‐Rad) and acquired with the ChemiDoc Touch Imaging System (Bio‐Rad). Signals were quantified with the Image Lab software (Bio‐Rad).
Cardiac troponin t
Manufactured by Abcam
Sourced in United States
Cardiac troponin T is a protein found in heart muscle cells. It is a key component of the cardiac troponin complex, which regulates the calcium-mediated interaction between actin and myosin, enabling muscle contraction. Cardiac troponin T is used as a biomarker to detect and monitor heart muscle injury or damage.
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Lab products found in correlation
Fluorescence microscope, by Olympus (2 mentions)
Nanog, by R&D Systems (2 mentions)
Karyomax giemsa stain stock solution, by Thermo Fisher Scientific (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
α actinin 2, by Merck Group (1 mentions)
Ripk3, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Er tracker green, by Thermo Fisher Scientific (1 mentions)
Grp78, by Abcam (1 mentions)
Oct3 4, by Santa Cruz Biotechnology (1 mentions)
Alpha actinin, by Abcam (1 mentions)
α fetoprotein, by Merck Group (1 mentions)
Cardiac troponin 1, by Abcam (1 mentions)
β tubulin 3, by Merck Group (1 mentions)
Tra 1 60, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
α sarcomeric actin, by Abcam (1 mentions)
14 protocols using cardiac troponin t
1
Western Blot Analysis of hiPSC-CMs
2
Immunofluorescence Staining of Cellular Markers
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After treatments, the samples were washed three times with PBS and were fixed with 4% paraformaldehyde for 30 min at room temperature [32] (link). Subsequently, the samples were incubated with the primary antibody at 4 °C overnight. Next, PBS was used to wash the samples three times, and subsequently the samples were stained with fluorescent second antibody at 37 °C for 30 min. DAPI was used for nuclear staining. The pictures were acquired using a fluorescence microscope (Olympus Corporation, Tokyo, Japan) with standard excitation filters. The following primary antibodies were used for immunofluorescence staining: Ripk3 (1:1000, Cell Signaling Technology, 95,702), GRP78 (1:1000, Abcam, ab21685), and Cardiac Troponin T (1:1000, Abcam, ab50576). Anti-mouse IgG (1:500, 4408, green), Anti-mouse IgG (1:500, 4409, red), anti-rabbit IgG (1:500, 4412, green) and anti-rabbit IgG (1:500, 4413, red) were purchased from Cell Signaling Technology. DAPI (Sigma-Aldrich, USA) and ER-Tracker™ Green (Molecular Probes, USA) were used to detect the nuclei and ER, respectively.
3
Immunostaining Undifferentiated and Differentiated Cells
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Undifferentiated cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100, blocked in 3% horse serum and stained for Nanog (R&D Systems, Abingdon, UK), Oct3/4 and Tra-1-60 (both from SantaCruz Biotechnology, Dallas, TX, USA). EBs differentiated to the three germ layers were fixed in cold methanol, permeabilized and blocked with 10% goat serum and stained for α-fetoprotein, β-tubulin III (both from Sigma-Aldrich, St. Louis, MO, USA) and Muscle actin (Dako, Glostrup, Denmark). Disaggregated beating bodies were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked in 3% goat serum and stained for cardiac troponin T, cardiac troponin I, and alpha actinin (all from Abcam, Cambridge, UK). Appropriate secondary antibodies were used and cells were counterstained and mounted with ProLong Gold Antifade Reagent with DAPI.
4
Immunofluorescence Analysis of Transplanted Cardiomyocytes
5
Cardiac Nuclei Ploidy Analysis by Imaging Flow Cytometry
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We used a FlowSight Imaging Flow Cytometer to combine the advantages of traditional flow cytometry and microscopy to verify the cardiac nuclei ploidy. Left ventricular cardiomyocyte nuclei were isolated and stained with Hoechst (Invitrogen, catalog no. H3570; 1:1,000) and the cardiomyocyte marker cardiac troponin T (Abcam, catalog no. ab56357; 1:250). The nuclei were resuspended in PBS at a concentration of 2 × 107 cells ml−1. The DNA content was detected by a FlowSight Imaging Flow Cytometer (Luminex), equipped with a ×20 objective lens, and analyzed by image analysis software (IDEAS). The percentage of 2n, 4n and greater than 4n ploidy was determined by setting gates using the calibration with nuclei of noncardiomyocytes at 2n. The software separated the cardiomyocyte nuclei using brightfield, Hoechst and Troponin T images.
Chromosomes were visualized by Giemsa staining (GIBCO KaryoMAX Giemsa Stain Stock Solution, catalog no. 10092–013) according to the manufacturer’s protocol. Briefly, sorted cardiac nuclei from 1465 were treated with a hypotonic solution (0.075 M KCl) and preserved in their swollen state with Carnoy’s fixative; further nuclei were dropped on to slides and air-dried. The slides were stained with freshly prepared Giemsa staining solution (3:1 ratio of Gurr Buffer and Giemsa Stain) and visualized at ×100 magnification.
Chromosomes were visualized by Giemsa staining (GIBCO KaryoMAX Giemsa Stain Stock Solution, catalog no. 10092–013) according to the manufacturer’s protocol. Briefly, sorted cardiac nuclei from 1465 were treated with a hypotonic solution (0.075 M KCl) and preserved in their swollen state with Carnoy’s fixative; further nuclei were dropped on to slides and air-dried. The slides were stained with freshly prepared Giemsa staining solution (3:1 ratio of Gurr Buffer and Giemsa Stain) and visualized at ×100 magnification.
6
Cardiomyocyte Differentiation Protocol
7
Immunofluorescence Characterization of Stem Cell Lineages
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ESCs and EB outgrowths were fixed with 3% PFA in PBS at room temperature for 10 min and then were permeabilized with 0.5% Triton-X 100 (Sigma–Aldrich) in PBS at room temperature for 5 min. Nonspecific antibody binding was blocked by incubation in 3% bovine serum albumin (BSA; Sigma–Aldrich) in PBS at room temperature for 1 h. Next, primary antibodies were diluted with 0.5% BSA in PBS and incubated with cells at 4°C overnight. Primary antibodies against the following epitopes were used: Oct-4 (Santa Cruz Biotechnology; diluted 1:100), Nanog (Cosmo Bio Co.; diluted 1:200), myosin heavy chain (DSHB, diluted 1:10), MyoD (Santa Cruz Biotechnology; diluted 1:200), and cardiac Troponin T (Abcam; diluted 1:100). The next day, specimens were incubated with appropriate secondary antibodies conjugated with Alexa 488 (Molecular Probes) or Alexa 595 (Molecular Probes) diluted 1:200 and DRAQ5 diluted 1:1,000 in 0.5% BSA in PBS at room temperature for 2 h. Specimens were washed in PBS and mounted using the Fluorescent Mounting Medium (Dako). The specificity of primary antibodies was confirmed by incubating ESCs and EB outgrowths with secondary antibodies alone. Specimens were analyzed using an Axiovert 100M scanning confocal microscope (Zeiss) equipped with LSM 510 software. Figures were assembled using Adobe Photoshop CS6 Extended.
8
Immunohistochemical Staining of Cell Markers
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High-temperature antigen retrieval and paraffin removal were performed by immersing slides in Trilogy (Cell Marque, Hot Springs, AR) in a pressure cooker until the chamber reached 126 °C and 23 psi. A Dual Endogenous block (Dako, Carpinteria, CA) was applied for 5 minutes. Slides were incubated with antibodies to Vimentin (Cell Signaling Technology, Danvers, MA), cardiac Troponin T (Abcam, Cambridge, MA), or CD31 (Thermo Fisher, Waltham, MA), followed by addition of a mouse or rabbit HRP detection system (Leica Biosystems, Chicago, IL). Finally, slides were stained with a DAB substrate (Vector Lab, Burlingame, CA) and counterstained with hematoxylin (Richard-Allen Scientific, Kalamazoo, MI).
9
Immunohistochemical Analysis of Ripk3 and Cardiac Troponin T
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After treatment, the samples were washed in phosphate-buffered saline (PBS) for 3 times and fixed using 4% paraformaldehyde at 25°C for 30 min. Then, the samples were incubated using Ripk3 (1:1,000 dilution, cat. no., 95702, Cell Signaling Technology, Danvers, MA, USA) and cardiac troponin T (1:1000, ab50576, Abcam, Cambridge, United Kingdom) antibodies at 4°C for 12 h. Subsequently, the samples were rinsed with PBS 3 times, followed by staining with fluorescent secondary antibody for 30 min at 37°C. DAPI was applied for nuclear staining. Fluorescence microscope (Olympus Corporation, Tokyo, Japan) was used for image taking.
10
Immunostaining of Cardiomyocytes and Heart Tissue
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Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked in 5% BSA for 1 h. Then cells were stained with the following primary antibodies overnight at 4°C: Cardiac troponin T (Abcam, #ab8295, 1:200), Samm50 (Proteintech, #28679-1-AP, 1:50), Pink1 (Proteintech, #23274-1-AP, 1:100) and VDAC1 (Santa Cruz Biotechnology, #sc-390996, 1:50). After washing with PBS 3 times, cells were incubated with specific secondary antibodies conjugated to Alexa Fluor for 1 h at 37°C. Five minutes after co-staining with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, #D1306), the cells were observed by a fluorescence microscope, and cardiomyocyte surface area was calculated by Image-Pro Plus software.
Heart sections were deparaffinized and rehydrated and then antigenically retrieved in sodium citrate buffer for 30 min. The following procedures were conducted according to the protocol described for cardiomyocytes. Co-localization was analyzed by Image-Pro Plus software.
Heart sections were deparaffinized and rehydrated and then antigenically retrieved in sodium citrate buffer for 30 min. The following procedures were conducted according to the protocol described for cardiomyocytes. Co-localization was analyzed by Image-Pro Plus software.
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