Cardiomyocytes were isolated from C57/B6 mice or Kit-CreER;Rosa26-RFP mice as described above. After obtaining the cell pellet, the collected cardiomyocytes were incubated with increasing concentrations of calcium ion in four steps. In details, cells were re-suspended with 0.5 mg/ml BSA in CaCl2 (0.06 mM, 0.24 mM, 0.6 mM and 1.2 mM)-containing MTS. Each step took 4 min. Cells were next re-suspended in 1.2 mM calcium chloride/10% FBS-containing alpha-MEM (Invitrogen) and plated on laminin (Invitrogen)-coated coverslips (Fisher or NEST) in 24-well cell culture plate (Corning) for at least 4 h. After well attached to the coverslips, cells were fixed in 4% PFA for 15 min and then blocked in blocking buffer (5% normal donkey serum in PBS with 0.5% triton X-100) for 30 min at room temperature. Subsequently, cells were incubated with primary antibodies (c-kit, R&D, AF1356, 1:50; TNNI3, Santa Cruz, sc-15368, 1:100; RFP, Rockland, 600401379, 1:1 000) at 4 °C for 24 h and then gently washed in PBS. Alexa Fluor fluorescent secondary antibodies (Invitrogen) were used to detect the signals. Following wash out of the secondary antibodies, the coverslips were mounted with fluorescence-protecting mounting medium (Vector Lab) and images were taken on an Olympus confocal microscopy system (FV1200).
Tnni3
Manufactured by Santa Cruz Biotechnology
TNNI3 is a laboratory reagent produced by Santa Cruz Biotechnology. It is a protein that plays a key role in the regulation of muscle contraction.
Automatically generated - may contain errors
Lab products found in correlation
Laminin, by Thermo Fisher Scientific (1 mentions)
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Alexa fluor fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fv1200, by Olympus (1 mentions)
24 well cell culture plate, by Corning (1 mentions)
Tnnt2, by Merck Group (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal fluorescence microscope, by Leica (1 mentions)
Goat serum, by Merck Group (1 mentions)
2 protocols using tnni3
1
Isolation and Immunostaining of Cardiomyocytes
2
Immunocytochemistry of Cardiomyocytes
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Cell sheets differentiated for 34 days were dissociated into single cells with TryPLE and seeded at low density onto LN-521+LN-221coated plates. After 3 days, CMs were washed once with PBS and fixed with 4% paraformaldehyde (PFA) (Sigma, Cat.#28908) for 20 mins at 4 C. The cells were then washed with PBS and blocked with blocking buffer containing 1% BSA, 5% goat serum (Sigma, Cat.#G9023) and 0.1% Triton X (Sigma, Cat.#T8787) for 1 hr at room temperature. After blocking, cells were incubated with primary antibody: TNNI3 (Santa cruz, Cat.#sc-15368, 1:20), NKX2-5 (Santa Cruz, Cat.#sc-14033, 1:100), TNNT2 (1:200), a-actinin (Sigma, Cat.#A7811, 1:500) in PBS containing 1% BSA and 5% goat serum at room temperature for 1 hour. Following this, antibody solution was removed and cells washed thrice with PBS on a shaker for 5 mins each. Subsequently, the cells were incubated at room temperature with Alexa-conjugated secondary antibody (ThermoFisher, 1:1000) on a shaker at low speed in the dark for 1 hr. Finally, cells were washed with PBS twice (5 mins) and ProLongâ Gold Antifade Mountant with DAPI (ThermoFisher, Cat.#P36931) added to stain the nuclei. Cells were visualized immediately in Leica TCS sp8 confocal fluorescence microscope.
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