Tyrosine hydroxylase th

The amygdala samples were harvested 24 h after final behavioral test. The amygdala of rats was ultrasonically disrupted in a cold radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology Co., Haimen, Jiangsu, China) with protease inhibitors (PMSF, Beyotime Biotechnology Co., Haimen, Jiangsu, China) followed by centrifugation at 12,000 × g for 20 min. Then, the total protein concentration of the supernatant was quantified by a BCA kit (Beyotime Biotechnology Co., Haimen, Jiangsu, China). Western blotting was performed as described previously (Hiroi et al., 1997 (link)). Primary antibodies used were tyrosine hydroxylase (TH, rabbit polyclonal, 1:2,500; Proteintech, Chicago, IL, United States); glial fibrillary acid protein (GFAP, rabbit polyclonal, 1:3,000; Abcam, MA, United States); Iba1 (rabbit monoclonal, 1:1,000; Abcam); DRD1 (rabbit monoclonal, 1:2,000; Abcam); DRD2 (rabbit polyclonal, 1:1,000; Abcam, MA, United States); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (rabbit monoclonal, 1:5,000; Abcam, MA, United States). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit (1:5,000; Abcam, MA, United States) and horseradish peroxidase-conjugated goat anti-mouse (1:5,000; Abcam, MA, United States).

Hearts were cut and fixed in 4% formalin for 48–72 h at 4°C. Sectioning was performed longitudinally (along the direction of blood flow). The segments of the peripheries of the myocardial scars and perivascular regions were processed into paraffin blocks, and oriented to clearly visualize the epicardial and endocardial surfaces. The paraffinized tissue blocks were cut into 3 μm-thick sections. For each paraffin block, one slide each was stained with hematoxylin-eosin (HE) to accentuate muscle and connective tissues. Then, immunohistochemical staining was performed to detect growth-associated protein 43 (GAP43), tyrosine hydroxylase (TH) and neurofilament (NF) (Proteintech Group, Rosemont, USA) and evaluate the nerve density according to standard procedures.
The density of the TH, NF, and GAP43 positive nerve fibers was assessed using the images of 20X magnification areas, which was obtained under a light microscope equipped with a computerized image system (Leica QWin V3, Germany). The density was expressed as the average of ten randomly selected fields (μm²/mm²). The average density of all the nerves in the field of view was regarded as the average density of nerves of the slices. Computer aided Bioanalysis Image Software 2012 (CEWEI Co., Ltd. Shanghai, China) was applied to calculate the density of the nerves.