The antibodies used for western blotting were: Phospho-Smad1/5 [Cell Signaling Technology (CST), 9516; RRID: AB_491015], Phospho-Smad2/3 (CST, 3108; RRID: AB_490941), Smad1 (CST, 9743; RRID: AB_2107780), Smad2/3 (CST, 5339; RRID: AB_10626777), Eomes (Abcam, ab23345; RRID:AB_778267), T (rabbit polyclonal anti-Brachyury, custom), Tcf3 (Santa Cruz Biotechnology, sc-166411), GAPDH (CST, 5174), H3 (Abcam, ab1791; RRID:AB_302613), active Ctnnb1 (CST, 8814; RRID: AB_11127203). The antibodies used for immunoprecipitation were: Tcf3 (Santa Cruz Biotechnology, sc-166411; RRID: AB_2302942), normal mouse IgG (Santa Cruz Biotechnology, sc-2025; RRID: AB_737182). The antibodies used for ChIP were: Phospho-Smad1/5/9 (CST, 13820; RRID: AB_2493181), Phospho-Smad2 (CST, 18338; RRID: AB_2798798), Eomes (Abcam, ab23345), T (Santa Cruz Biotechnology, sc-17743; RRID:AB_634980). The antibodies used for immunofluorescence and flow cytometry were: Myl7 (Proteintech, 17283-1-AP; RRID: AB_2250998; 1:250 dilution), Actc1 (Proteintech, 66125-1-Ig; RRID: AB_2881524; 1:200), Tnnt2 (Abcam, ab209813; 1:200), donkey anti-mouse IgG, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202; RRID: AB_141607; 1:500), donkey anti-rabbit IgG, Alexa Fluor 488 (Thermo Fisher Scientific, A-21206; RRID: AB_2535792; 1:500).
Tnnt2
Manufactured by Abcam
Sourced in United States
TNNT2 is a protein that plays a crucial role in the contraction of cardiac muscle. It is a component of the thin filament in the sarcomere, the basic contractile unit of muscle. TNNT2 is involved in the regulation of muscle contraction by interacting with other proteins in the sarcomere.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Nanog, by Santa Cruz Biotechnology (2 mentions)
α actinin, by Abcam (2 mentions)
Ssea4, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Nis elements ar software, by Nikon (2 mentions)
Fitc conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Tripsin edta, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Sangon (2 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Actc1, by Proteintech (1 mentions)
Eomes, by Abcam (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Axio observer a1 fluorescence microscope, by Zeiss (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Alp substrate kit 3, by Vector Laboratories (1 mentions)
10 protocols using tnnt2
1
Antibody Selection for Pluripotency and Lineage Analysis
2
Immunofluorescent Staining of Pluripotency and Cardiac Markers
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Cells were fixed with 4% paraformaldehyde (PFA) (Sangon Biotech) for 15 minutes, permeabilized with 0.1% Triton X (Sangon Biotech) for 5 minutes, and blocked with 3% BSA (Sigma‐Aldrich) for 1 hour. Cells were subsequently stained with appropriate primary antibodies and AlexaFluor conjugated secondary antibodies (Life Technologies). Nuclei were stained with DAPI (Roche Diagnostics). For the staining of pluripotency markers, the primary antibodies were OCT4 (Santa Cruz Biotechnology), NANOG (Santa Cruz Biotechnology), SSEA‐4 (Abcam) and SOX2 (Abcam). For the staining of cardiac‐specific markers, the primary antibodies were TNNT2 (Abcam) and α‐actinin (Abcam). Pictures were taken with 63× objective on confocal microscope (Nikon, A1) using NIS‐Elements AR software (Nikon).
3
Stem Cell Characterization Protocol
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Alkaline phosphatase (ALP) activity was analysed by using an ALP substrate kit III (Vector Laboratories, USA) according to the manufacturer's instructions. Immunostaining assays were performed according to the protocol described previously [24 (link)]. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100, blocked in 10% normal goat serum (Vector Laboratories), and then incubated with primary antibodies against OCT4 (1 : 200, Abcam, USA), NANOG (1 : 200, Abcam, USA), and TNNT2 (1 : 500; Abcam, USA) in 4°C overnight and detected by DyLight 488- or DyLight 549-conjugated secondary antibodies. Nuclei were stained with DAPI (Sigma, USA). A Zeiss Axio Observer A1 fluorescence microscope was used for slide observing and image capture.
4
Immunofluorescence Staining of Cells
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Cells were fixed with 2% v/v paraformaldehyde (EMS) as described previously (32 (link)). Fixed cells were blocked in antibody buffer (5% w/v BSA, 0.1% v/v Tween-20, in PBS) for 1 h at room temperature. Following blocking, cells were incubated overnight at 4 °C with a targeting antibody (Epas1: Novus Biological Cat no NB100-122; Tnnt2: Abcam Cat no ab45932) in antibody buffer. After the overnight incubation, cells were washed three times in an antibody buffer. Following washing, cells were incubated with Alexa-Fluor conjugated secondary antibodies (ThermoFisher) at a 1:500 dilution in antibody buffer for 1 h at room temperature. Nuclei were stained by DAPI at 1 μg/ml for 30 min at room temperature in antibody buffer. Phalloidin was used per manufacturers’ instructions (ThermoFisher). Following washing in PBS to remove unbound complexes, immunofluorescence was measured using a Zeiss Axiovert 200 inverted microscope.
5
Cardiomyocyte Protein Expression Analysis
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The iPSC-CMs were grown in 6-well plates to 80% confluence, detached with TrypLE (Gibco), and then pelleted at 10,000 rpm for 3–5 min at 4 °C. The pellets were washed twice with Dulbecco’s Phosphate-Buffered Saline (DPBS) (Gibco) and then lysed in RIPA (Solarbio). Lysates were placed on ice for 30 min and subsequently centrifuged at 12,000 rpm for 15 min to collect the supernatants. Protein concentration was measured using a BCA kit (Thermo Fisher Scientific). Protein was separated in 12% protein precast gels (Genscript) and transferred to a PVDF membrane. Block the membrane in 5% skimmed milk in Tris-Buffered Saline and Tween 20 (TBST) for 1 h, incubate overnight in 4 °C with primary antibodies, and then incubate with secondary antibodies (Cell Signaling Technology) at room temperature for 1.5 h. Primary antibodies include Kir2.1 (Proteintech), Nav1.5 (Alomone Labs), RYR2 (Abcam), TNNT2 (Abcam), MYBPC3 (Santa Cruz Biotechnology), KCNH2 (Santa Cruz Biotechnology), Cav1.2 (Abcam) and GAPDH (Abmart).
6
Quantitative Analysis of TNNT2 Protein in Zebrafish
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Total protein of compound-treated zebrafish was extracted with the Tissue Protein Rapid Extraction Kit (Keygen, China) and determined by performing the bicinchoninic acid (BCA) protein assay (Pierce, Waltham, MA). The equal amounts of 100 mg lysate proteins were loaded onto SDS-polyacrylamide gels (12%) at 80 V and electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA) in Tris-glycine buffer at 100 V in an ice box. After blocking with 5% nonfat milk in TBST for 1 h at room temperature, the membrane was incubated with TNNT2 (1:1000, rabbit antibodies, Abcam, London, UK) overnight at 4 °C, After washed thrice with TBST, the membrane was incubated with a horseradish peroxidase-conjugated anti-rabbit Ig G secondary antibody (Abcam, Britain) for 1 h on a shaking table at room temperature, and washed thrice with TBST and the antibody-bound proteins were detected using the ECL chemiluminescence reagent (Pierce, Waltham, MA).
7
Immunostaining of Dissociated iPSC-CMs
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Monolayer iPSC-CMs were dissociated into single cells using 0.25% Tripsin-EDTA (Gibco) for 5 min at 37 °C. Cells were pelleted and fixed with 2% paraformaldehyde (PFA) (Sangon Biotech) for 10 min on ice. Every step was washed with 5% fetal bovine serum (Gibco) in phosphate-buffered saline (PBS) (Sangon Biotech) before sample centrifugation. Cells were stained with TNNT2 (Abcam) at 4 °C. FITC-conjugated goat anti-mouse IgG antibody (Invitrogen) was used as the secondary antibody.
8
Immunofluorescence Staining of Pluripotency and Cardiac Markers
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Cells were fixed with 4% PFA for 10 minutes, permeabilized with 0.1% Triton X (Sangon Biotech) for 10 minutes at room temperature and blocked with 3% BSA (Sigma-Aldrich) for 1 hour. Cells were then washed and subsequently stained with the appropriate primary antibodies and AlexaFluor conjugated secondary antibodies (Life Technologies). Nuclei were stained with DAPI (Roche Diagnostics) or Hoechst (Solarbio, HOE 33258). The primary antibodies of OCT4 (Santa Cruz Biotechnology), NANOG (Santa Cruz Biotechnology), SSEA-4 (Abcam), and SOX2 (Abcam) were stained as pluripotency markers. The primary antibodies of TNNT2 (Abcam) and α-actinin (Abcam) were stained as cardiac-specific markers. The NR2F2 (Abcam) and MLC2a (Abcam) were staining as atrial-specific markers. The α-tubulin in microtubule network was stained with Tubulin-TrackerTM Deep Red (Sigma-Aldrich, T34077) and β-tubulin was stained with Tubulin-Tracker green staining kit (Beyotime, C22135). Pictures were obtained with 60× objective on confocal microscope (Nikon, A1) using NIS-Elements AR software (Nikon).
9
Apoptosis Quantification of hESC-CMs
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Apoptosis of hESC‐CMs was measured using an In‐Situ Cell Death Detection Kit (Roche Diagnostics) in accordance with the manufacturer's instructions. Cells were co‐stained with TNNT2 (Abcam) as described above. Images were collected and analysed using an inverted microscope (Nikon, Eclipse Ti‐S).
10
Dissociation and Fixation of Cardiomyocytes
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Monolayer CMs were dissociated into single cells using 0.25% Tripsin‐EDTA (Gibco) for 5 minutes at 37°C. Cells were pelleted and fixed with 4%PFA (Sangon Biotech) for 10 minutes on ice. Every step was washed with PBS (Sangon Biotech) before sample centrifugation. Cells were stained with TNNT2 (Abcam) at 4°C, and FITC‐conjugated goat anti‐mouse IgG antibody (Invitrogen) was used as secondary antibody.
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