Cells were fixed with phosphate buffer saline (PBS) containing 4% paraformaldehyde at room temperature for 30 min. After washing with PBS, cells were permeated with 0.05% Triton X-100 (Sigma-Aldrich) for 15 min, then blocked with 1% BSA (Sigma-Aldrich) plus 4% normal goat serum (Sigma-Aldrich) at room temperature for 1 h. The cells were incubated with primary antibodies overnight at 4°C: anti-octamer-binding transcription factor 4 (Oct-4, dilution 1:100, Santa Cruz, Dallas, TX, USA), anti-cardiac troponin I (cTn I, dilution 1: 200, Abcam, Cambridge, MA, USA), anti-α-myosin heavy chain (α-MHC, dilution 1:100, Abcam). After washing with PBS, The cells were incubated with secondary antibodies (dilution 1: 500, Abcam) at room temperature for 1 h and then counterstained with 4′6-diamidino-2-phenylindole (DAPI, dilution 1: 1,000, Sigma-Aldrich) for 5 min. Samples were examined using Olympus FV1000 confocal laser scanning microscope (Olympus, Japan). For manual quantification, samples from each group were examined for α-MHC and cTn I+ cells in 5 random fields with each field contained 50 cells.
Anti α myosin heavy chain α mhc
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-α-myosin heavy chain (α-MHC) is a lab equipment product that is used to detect the presence and/or quantity of the α-myosin heavy chain protein. This protein is a component of the myosin enzyme complex, which is responsible for muscle contraction.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Anti cardiac troponin 1 ctn 1, by Abcam (1 mentions)
Cytoflex cytometer, by Beckman Coulter (1 mentions)
Fix and perm kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Cytexpert acquisition and analysis software, by Beckman Coulter (1 mentions)
Anti cardiac troponin t ctnt, by Abcam (1 mentions)
Isotypes, by BD (1 mentions)
2 protocols using anti α myosin heavy chain α mhc
1
Immunofluorescence Analysis of Cardiac Markers
2
Characterization of Cardiomyocyte Markers
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For flow cytometry and imaging flow cytometry, cells were treated with the FIX and PERM® Kit (Thermo Fischer Scientific, Waltham, MA, USA) and incubated for 1 h at RT with anti-α-myosin heavy chain (α-MHC; 1:100; Abcam, Cambridge, UK), anti-cardiac troponin T (cTnT, 1:100; Abcam, Cambridge, UK), anti-α-sarcomeric actin (α-SA; 1:100; Abcam, Cambridge, UK), anti-sarcolemmal Ca2+ channel (CACNA1C, 1:100; Abcam, Cambridge, UK), and anti-sarcoendoplasmic reticulum Ca2+ ATPase protein (Serca2, 1:100; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), followed by incubation with the appropriate secondary antibody conjugated with Alexa Fluor 488, PE-Alexa Fluor 647, or PE-Cy7 (1:200; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), as previously reported [53 (link)]. Cells incubated with isotypes (all from BD) were used as negative controls [54 (link)].
Cytometric analysis was performed with a Cytoflex cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) or CytExpert Acquisition and Analysis Software (Beckman Coulter, Brea, CA, USA).
Cytometric analysis was performed with a Cytoflex cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) or CytExpert Acquisition and Analysis Software (Beckman Coulter, Brea, CA, USA).
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