Gtx127937

Heart tissue was cut into 7-μm-thick sections and fixed with acetone at 4°C for 10 min. Next, the sections were incubated with rabbit polyclonal anti-GAP43 (GTX127937; GeneTex, 1:200) and sheep polyclonal anti-tyrosine hydroxylase (TH) (AB1542; Millipore, 1:400) primary antibodies overnight at 4°C followed by incubation with Alexa 488-conjugated donkey anti-sheep (A-11015; Invitrogen, 1:400) and Alexa 594-conjugated donkey anti-rabbit (A-21207; Invitrogen, 1:400) secondary antibodies for 2 h at room temperature. Then, the cell nuclei were stained with DAPI (ab104139; Abcam, UK). Finally, the sections were blocked with an anti-fluorescence quencher. The areas of GAP43 and TH expressions were calculated using the ImageJ software (version 1.8).

Proteins were extracted from the heart tissue, and the concentrations were measured using a BCA protein colorimetric assay kit (E-BC-K165-M; Elabscience, China). The protein samples were separated on 12.5% SDS polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (IPVH00010; Millipore, MA, USA). The membranes were blocked for 1 h in 5% non-fat milk diluted in TBS and then incubated with rabbit polyclonal anti-growth-associated protein 43 (GAP43) (GTX127937; GeneTex, 1:5,000) and rabbit monoclonal GAPDH (5174; Cell Signaling Technology, 1:1,000) primary antibodies overnight at 4°C and subsequently with a goat anti-rabbit IgG-HRP (abs20040; Absin, 1:5,000) secondary antibody for 1 h at room temperature. Protein bands were detected using an ECL chromogenic substrate (WBKLS0500, Millipore, USA) and analyzed using a chemiluminescence apparatus (Bio-Rad, USA).