Ti u fluorescent microscope

Myocytes were isolated from adult rats 37–45 weeks after sham or PO surgery. Briefly, adult rat hearts were heparinized, digested with collagenase and hyaluronidase [29 (link)]. Calcium-tolerant, isolated cells were resuspended to 4 × 10³ rod-shaped myocytes/mL in DMEM supplemented with 5% FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin (P/S) [29 (link)]. For adhesion studies, 100 µL myocyte aliquots were plated in triplicate onto 12 mm laminin-coated (0–40 µg/mL), glass coverslips. After 30 min, cells were gently washed twice with serum-free M199 media supplemented with P/S. Myocytes remaining attached to the coverslip after media replacement were counted under a light microscope to evaluate cell adhesion.
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.

Cultured primary neurons were fixed in cold acetone/methanol for 10 minutes. After permeabilization and blocking with 0.1% Triton X100 in PBS containing 1% BSA for 1 hour, the neurons were then incubated with primary antibody against CAPON (rabbit pAb, 1:200; Abcam) and tyrosine hydroxylase (mouse mAb, 1:200; Sigma) in 1% BSA overnight at 4°C. Signals were visualized with antirabbit antibody conjugated to Alexa-488 (1:1000; Molecular Probes) and antimouse antibody conjugated to Alexa-594 (1:1000; Molecular Probes). Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000; Sigma). Imaging was performed on a Nikon Ti-U fluorescent microscope.