Another subset of sham and pressure-overloaded myocytes were resuspended in DMEM plus 5% FCS and P/S (1 × 105 rod-shaped cells/mL), and plated on 25 mm2 laminin-coated coverslips [36 ]. Serum-free M199 media supplemented with P/S was added after 2 h. Myocytes were then fixed in 3% paraformaldehyde and immunostained with anti-nid-1 primary and Texas Red conjugated secondary Abs [21 (link), 29 (link)]. Myocytes were imaged with a Nikon Ti-U fluorescent microscope equipped with a DS-U2 digital camera and NIS Elements software. Some myocytes also were collected in ice-cold sample buffer for nid-1 analysis using SDS-PAGE and Western blots, as described earlier.
Isolation and Culture of Adult Rat Cardiomyocytes
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Corresponding Organization : University of Michigan–Ann Arbor
Other organizations : Korea Basic Science Institute
Protocol cited in 2 other protocols
Variable analysis
- Sham or pressure overload (PO) surgery on adult rats
- Myocyte adhesion to laminin-coated coverslips
- Nidogen-1 (Nid-1) immunostaining and expression in myocytes
- Calcium-tolerant, rod-shaped myocytes isolated from adult rat hearts
- Myocytes resuspended in DMEM supplemented with 5% FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin (P/S)
- Laminin-coated coverslips used for cell adhesion and immunostaining experiments
- Not explicitly mentioned
- Not explicitly mentioned
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