Ab41528

Right renal arteries with perivascular tissues were cut into three segments: proximal (close to aorta), middle, and distal (close to kidney). The arterial segments were fixed, embedded in paraffin, and cut into sections of 5μm in length. Every 5^th section was collected and 20 sections from each segment were stained with immunohistochemistry for tyrosine hydroxylase (TH). Therefore, 60 sections from each rat were analyzed. The immunohistochemistry staining for TH was performed using rabbit anti-TH antibody (dilution: 1:200, ab41528, Abcam, Cambridge, UK) [27 (link)]. After being washed, sections were incubated with goat anti-rabbit immunoglobulin G biotinylated secondary antibody (dilution: 1:200; SA1022, Boster, Wuhan, China) for 1 h at room temperature, and then detected using a DAB kit (AR1000, Boster, Wuhan, China). Sections were examined with an Olympus BX41 microscope (Olympus Corp., Tokyo, Japan). The TH-positive area (brown) and the entire area of the sympathetic nerves in each section were measured using ImageJ software and used to calculate the percentage of TH-positive area out of the entire sympathetic nerve area.

Thirty days after reperfusion, pigs were euthanized with potassium chloride (40 mEq/kg, I.V.). Tissues, including renal arteries and hearts, were collected for histological, biochemical, and molecular analyses. The renal arteries and surrounding tissues were subjected to immunohistochemical staining for hematoxylin and eosin (HE) and tyrosine hydroxylase (TH, Abcam, ab41528, 1:1000). Briefly, renal artery segments were harvested and fixed in 4% paraformaldehyde for 24 h, washed and placed in 70% ethyl alcohol until tissue processing for paraffin embedding. Renal artery samples were embedded and sliced into 2 μm thick serial cross-cryosections. LV tissue isolated from the infarct zones (IZs), BZs, and RZs was cut into 1 cm sections and 5 μm cross-sections were mounted onto glass slides and stained with HE and Masson’s trichrome staining for analysis of the infarct area and fibrosis. CD163 (Proteintech, 16646-1-AP, 1:200) were stain for inflammatory cells under established protocol.