Rabbit anti-SORBS2 (1:100; Proteintech) and mouse anti-Sarcomeric Alpha Actinin (1:100, Abcam);mouse and rabbit monoclonal anti-β-tubulin (1:100; CST); rabbit polyclonal anti-RyR2 (1:200; Invitrogen); rabbit polyclonal anti-JP2 (1:100; Abcam); rabbit polyclonal anti-Nanog (1:200;CST); rabbit monoclonal anti-Oct4A (1:100;CST); rabbit polyclonal anti-MLC-2V (1:100; proteintech); rabbit polyclonal anti-CTNI (1:100; Proteintech); rabbit polyclonal anti-CTNT (1:100; Proteintech); rabbit polyclonal anti-Sox2 (1:100; CST); rabbit polyclonal anti-SSEA (1:100; CST); Alexa 488-conjugated goat anti-mouse (Yeasen Biology); Alexa 594-conjugated goat anti-rabbit (Yeasen Biology). The cell nuclei were counterstained with 0.1% 4′, 6-diamidino-2-phenylindole (DAPI).
Anti mlc 2v
Manufactured by Proteintech
Sourced in United States
Anti-MLC-2V is a rabbit polyclonal antibody that recognizes Myosin Light Chain 2, Ventricular/Cardiac (MLC-2V) protein. It is commonly used for the detection and analysis of MLC-2V expression in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti mlc2a, by Synaptic Systems (4 mentions)
Anti ctni, by Proteintech (2 mentions)
Hoechst 33342, by Dojindo Laboratories (2 mentions)
Anti α actinin, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Harmony analysis software, by PerkinElmer (1 mentions)
Operetta high content imaging system, by PerkinElmer (1 mentions)
Fluorescent mounting solution, by Agilent Technologies (1 mentions)
Confocal fluorescence microscope, by Zeiss (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti ctnt, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Anti ctnt, by Bioss Antibodies (1 mentions)
Anti α sma, by Boster Bio (1 mentions)
Anti cd31, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fibronectin coated 96 well plate, by Corning (1 mentions)
8 protocols using anti mlc 2v
1
Multimarker Immunofluorescence Staining Protocol
2
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).
3
Immunofluorescence Staining of Cardiac Markers
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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).
4
Cardiac Protein Expression Analysis
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Cardiac proteins (DHPR, RyR2, cTnI, Connexin45, MLC-2v, cTnT and cMHC) were also detected by flow cytometry. The cells after 3 days of culture (no differentiation at this time) or after appearance of first beating were digested with 0.25% trypsin, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated for 1 h at 4°C with anti-DHPR (1∶50), anti-RyR2 (1∶50), anti-cMHC (1∶100), anti-cTnI (1∶100), anti-Connexin45 (1∶100), anti-MLC-2v (1∶100; Proteintech, Chicago, IL, USA), and anti-cTnT (1∶100; Beijing Biosynthesis Biotechnology) specific primary antibodies. The cells were then incubated with FITC (Beijing Biosynthesis Biotechnology) and phycoerythrin/PE (Proteintech) conjugated secondary antibodies for 1 h at 4°C. Finally, cells were washed with PBS and resuspended in 4% paraformaldehyde for flow cytometry. The negative control tubes were not incubated with primary antibody but with fluorescently-labeled second antibody. Data were analyzed by the FlowJo and DiVa package (Tree Star, Inc. Ashland, OR, U.S.A).
5
Cardiac Cell Phenotyping Using Immunofluorescence
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Heart sections were stained with anti-MLC2V (Proteintech; 1:50), anti-α-SMA (BOSTER; 1:50) and anti-CD31 (Abcam, 1:50) at 4 °C overnight. The samples were washed with PBS and then incubated with secondary antibodies at room temperature for 1 h. DAPI was used for nuclear staining. The immunofluorescence-stained sections were imaged with a Zeiss confocal microscope.
6
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.
7
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).
8
Immunostaining of Micropatterned iPSC-CMs
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Dissociated iPSC-CMs differentiated for ∼40 days were seeded at single-cell density on glass coverslips micro-patterned with 20-μm-wide gelatin lines as described previously (Birket et al., 2015 (link)). Four days later cells were fixed, permeablized, and labeled. Antibodies were as follows: anti-α-actinin (Sigma; A7811), anti-MLC-2V (Proteintech; 10906-1-AP), and anti-myomesin-3 (Proteintech; 17692-1-AP). Individual separated CMs were identified based on α-actinin labeling and then MLC-2V or myomesin-3 labeling was also imaged and recorded. Cell area was calculated using a thresholding mask based on α-actinin labeling.
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