Rabbit anti ngf

Samples of myocardial tissues from the infarcted border zone were prepared for protein analysis. The heart tissues samples were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail. Protein concentrations were determined using the BCA kit (Beyotime, Shanghai, China), and 40 μg protein lysates were electrophoresed and separated on 10–15% SDS-PAGE before transfer onto PVDF membranes (Millipore, Boston, USA). The membranes were blocked with 5% skim milk for an hour at room temperature and then incubated overnight at 4°C with primary antibodies, namely, rabbit anti-NGF (1 : 1000, Abcam, UK), rabbit anti-TrKA (1 : 1000, Abcam, UK), rabbit anti-tyrosine hydroxylase (1 : 10000, Abcam, UK), rabbit anti-p-PI3K (1 : 1000, affinity, USA), rabbit anti-p-Akt (1 : 1000, Cell Signaling Technology, USA), and rabbit anti-p-Bad (1 : 1000, ImmunoWay, USA). The membranes were washed thrice with Tris-Buffered Saline Tween, followed by incubation with the horseradish peroxidase-conjugated secondary antibody (1 : 5000) for 2 hours at RT. Antigen-antibody complexes were then visualized using an enhanced chemiluminescence kit (Applygen, Beijing, China), and relative band densities of proteins in the western blots were normalized against GAPDH.

Nasal mucosa was fixed with 4% paraformaldehyde, and then dehydrated. After that, nasal mucosa was cut into sections (4 μm) following paraffin embedding. Slices were baked and dewaxed to water before incubated at room temperature with 3% hydrogen peroxide for 10 min. Subsequently, the slices were blocked in 10% goat serum and 0.3% Triton X-100 PBS solution for 1 h, followed by incubated overnight with corresponding antibodies (4 °C), and then IL-4 and IFN-gamma were double stained with mast cell markers. The primary antibodies and mast cell markers were rabbit anti-NGF (abcam, 1:100), rat anti-IL-4 (Santa, 1:200) and rabbit anti-Mast Cell Chymase (MCC, affinity, 1:100), rabbit anti-IFN-gamma (affinity, 1:100) and mouse anti-Mast Cell Tryptase (MCT, Santa, 1:200). The secondary antibody corresponds to the primary antibody. The nuclei were stained with DAPI. NGF, IL-4 and IFN-gamma positive cells in nasal mucosa sections were observed by fluorescence microscopy (FluoView FV1000, Olympus). Image Pro Plus 6 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze positive cells.

At 3, 14, and 28 days after initiation of the exercise regimens, rats were sacrificed for Western blot analysis. Tissue samples from the ipsilesional ischemic cerebral hemispheres of all experimental groups were harvested, and total protein extraction was performed using cell lysis solutions (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein concentration was then determined by the BCA method. Electrophoresis (10% SDS-PAGE gel) was performed with 30 μg of protein per lane. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk, followed by incubation with primary antibodies (1:1,000 rabbit anti-BDNF, rabbit anti-NGF, rabbit anti-PSD-95, rabbit anti-SYN, rabbit anti-Tau, and rabbit anti-GAP43, Abcam, MA, USA; 1:500 rabbit anti-HIF-1α, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The next day, membranes were washed three times and further incubated with a goat anti-rabbit IgG-HRP secondary antibody (1:1,000, Santa Cruz) at room temperature for 1 h. After washing, the ECL method was used to detect signals. Western blot images for each antibody were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health, Bethesda, MD, USA) to quantify protein expression according to relative image density.