CMs were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde dissolved in PBS for 20 min. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min, washed in PBS + 0.1% Tween 20 (PBST), and blocked with 5% normal goat serum (NGS, Thermo Fisher Scientific) in PBST. The cells were stained with the following primary antibodies: anti-cTnT (Thermo Fisher Scientific), anti-α-actinin (Sigma-Aldrich), anti-MLC2v (Proteintech, Rosemont, IL, USA), anti-MLC2a (Synaptic Systems, Goettingen, Germany), and anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA, USA) antibodies at 4 °C overnight in 2% NGS in PBST. The cells were washed twice in PBST and incubated for 1 h with the following secondary antibodies: Alexa Fluor 488 anti-mouse IgG1, Alexa Fluor 594 goat anti-mouse IgG2b, and Alexa Fluor 594 goat anti-rabbit IgG (all from Molecular Probes, Eugene, OR, USA). The nuclei were stained with DAPI, and the stained cells were mounted using a fluorescent mounting solution (DAKO, Carpinteria, CA, USA). Immunofluorescence images were acquired using a fluorescence microscope (Olympus-Europa GmbH, Hamburg, Germany) and a confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Anti mlc2a
Manufactured by Synaptic Systems
Sourced in United States, Denmark
Anti-MLC2a is a laboratory reagent used in research applications. It is a specific antibody that targets the MLC2a protein, which is involved in muscle contraction. The primary function of Anti-MLC2a is to facilitate the detection and analysis of MLC2a in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti mlc 2v, by Proteintech (4 mentions)
Hoechst 33342, by Dojindo Laboratories (2 mentions)
Alexa fluor 488 anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Confocal fluorescence microscope, by Zeiss (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti α actinin, by Merck Group (1 mentions)
Fluorescent mounting solution, by Agilent Technologies (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti ctnt, by Thermo Fisher Scientific (1 mentions)
Anti collagen type 1, by Abcam (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Anti collagen type 3, by Abcam (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
Anti vimentin, by Agilent Technologies (1 mentions)
Anti laminin, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti connexin 43, by Abcam (1 mentions)
Anti sarcomeric alpha actinin α actinin, by Merck Group (1 mentions)
Anti cardiac troponin t ctnt, by Abcam (1 mentions)
Operetta high content imaging system, by PerkinElmer (1 mentions)
5 protocols using anti mlc2a
1
Immunofluorescence Staining of Cardiac Markers
2
Immunofluorescence Characterization of hiPSC-Derived Cardiomyocytes
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hiPSC-CMs and 3D-hiPSC-CT were fixed with 4% paraformaldehyde. hiPSC-CMs and 3D-hiPSC-CT were labeled by primary antibodies such as anti-cardiac troponin T (cTnT, 1:200 dilution; Abcam, Cambridge, UK), anti-sarcomeric alpha actinin (α-actinin, 1:400; Sigma), anti-vimentin (1:100; Dako, Glostrup, Denmark), anti-connexin43 (1:100; Abcam), anti-MLC2a (1:100; Synaptic Systems GmbH), anti-MLC2v (1:200; Proteintech, Rosemont) anti-fibronectin (1:200; Abcam), anti-laminin (1:30; Sigma), anti-collagen type I (1:100; Abcam), or anti-collagen type III (1:100; Abcam), followed by secondary antibodies such as AlexaFluor488 or AlexaFluor555 conjugated goat or donkey anti-mouse or anti-rabbit (ThermoFisher Scientific). Nuclei were counterstained with Hoechst33342 (Dojindo, Kumamoto, Japan) and assessed using confocal microscope (FLUOVIEW FV10i; Olympus, Tokyo, Japan).
3
Immunofluorescence Characterization of hiPSC-Derived Cardiomyocytes
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hiPSC-derived cardiomyocytes were fixed with 4% PFA and permeabilized with 0.1% Triton-X in PBS (-) for 15 min at 4°C. Then, the cells were blocked with 5% BSA in PBS (-) for 60 min at room temperature. Primary antibodies were reacted for 24 h at 4°C, and secondary antibodies were reacted for 1 h at room temperature. Nuclei were labeled with Hoechst 33342 (Dojindo, H342). Primary antibodies were anti-Troponin T (clone 13-11) (1:200, Thermo Scientific, MA5-12960), anti-MLC2a (1:200, Synaptic Systems, 311 011), and anti-MLC2v (1:200, ProteinTech, 10906-1-AP). Secondary antibodies were Alexa Fluor 488, donkey anti-mouse IgG (HCL), Alexa Fluor 568, donkey anti-rabbit IgG (HCL), Alexa Fluor 647, donkey anti-mouse IgG (HCL). Fluorescence images were obtained using Operetta high content imaging system (PerkinElmer, Japan) and analyzed using Harmony analysis software (PerkinElmer, Japan).
4
Flow Cytometry Profiling of hPSC-Derived Cardiomyocytes
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hPSC-derived cardiomyocytes were trypsinized and fixed in 1% formaldehyde at room temperature for 20 min. Cells were washed and incubated in PBS + 1% FCS with anti-cTnT (ab45932, Abcam) and anti-Mlc2a (311011, Synaptic Systems) 1:200 overnight at 4 °C. Subsequently, cells were stained with secondary antibodies (Alexa Fluor® 488 or 555, Invitrogen) 1:200 for 30 min on ice. After washing, cells were resuspended in 1× PBS + 1% FCS and analyzed on a BD FACSCanto™ II flow cytometer.
5
Cardiac Differentiation Protein Analysis
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AIC- and VIC-CD151high/low CMs were sorted and then seeded at 1 × 105 cells/well on a fibronectin-coated 96-well plate (Corning). The cells were fixed after 5–7 d of culture for 20 min with 4% paraformaldehyde, permeabilized for 15 min with 0.1% TritonX100-PBS, and then blocked for 1 h at room temperature with 2% goat serum/0.1% TritonX100-PBS. The fixed cells were stained using anti-MLC-2A (Synaptic systems, 1:100) and anti-MLC-2V (Proteintech, 1:200) overnight at 4 °C. The following day, the cells were washed twice with PBS and stained using Alexa Fluor 647 goat anti-mouse IgG (Thermo Fisher Scientific, 1:500), Alexa Fluor 647 goat anti-rabbit IgG (Thermo Fisher Scientific, 1:500), or Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific, 1:500) as the secondary antibody for 1 h at 4 °C under dark conditions. The cells were washed twice with PBS and stained with Hoechst (DOJINDO) for 5 min at room temperature. Images were captured using a BZ-X710 (KEYENCE) with a 20× objective.
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