4 6 diamino 2 phenylindole dapi

Adiponectin peptide was entrusted and synthesized by Sangon Biotech Co., Ltd (Shanghai, China). Glutamate, dichlorofluorescin diacetate (DCF-DA), and 4′,6-diamino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Beyotime (Shanghai, China). The assay kits of LDH, MDA, SOD, and GSH-Px were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kits were purchased from Roche (Mannheim, Germany). Antibodies against SIRT1, cleaved caspase-3, Bax, Bcl-2, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against PGC-1α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The appropriate secondary antibodies were purchased from Beyotime (Shanghai, China). SIRT1 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

A549 investigated cells were fixed in 3.8% paraformaldehyde solution containing 0.2% Triton X-100 (Sigma-Aldrich, USA) for 5 min at 37°C and subjected to immunostaining. To evaluate the effect of angiotensin-(1–7) and miRNA transfections in A549 cellular morphology, the actin filaments were stained in a 1% bovine serum albumin (BSA) solution containing 100 μg/ ml of phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) (Sigma-Aldrich, USA) for 1 h. Next, the nuclei of the cells were counterstained in 3.33 ng/ ml 4’,6 diamino-2-phenylindole (DAPI, Sigma-Aldrich, USA) solution for 5 min. After extensive washes with saline phosphate buffer (PBS, 2.7 mM KCl, 1.5 mM KH₂PO₄, 137 mM NaCl, and 8 mM Na₂HPO₄, pH 7.4), the coverslips containing cells were subsequently mounted onto slides and subjected to microscopic immunofluorescence analysis. Images were obtained with an Olympus BX51 microscopy equipped with corresponding filter sets and a DP71 CCD camera (Tokyo, Japan). One hundred randomly selected cells were analyzed in each investigated group. For phase-contrast analyses required in the in vitro scratching assays (wound healing) and in the cellular invasion chamber assays, the same microscopy was used and images were monitored and quantified using the Image J software (http://rsb.info.nih.gov/ij/).

The paraffin sections of human or mouse skin were flushed with PBS, then sealed with 5% donkey serum containing 0.3% Triton-100 at room temperature for 2 h. After blocking, anti-CD68 (1 mg/mL diluted by 1:100), anti-TLR2 (0.6 mg/mL diluted by 1:100), anti-KLK5 (1.295 mg/mL diluted by 1:100) and anti-IL-6 (0.555mg/ml diluted by 1:100) antibodies (all from Abcam) were incubated overnight at 4°C in a dark wet box. The sections were then incubated with Alexa Fluor 488 or 555 antibodies as the secondary antibody at room temperature for 2 h. Finally, 4′, 6-diamino-2-phenylindole (DAPI, Sigma) was counterstained for 15 min.
RAW 264.7 cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with PBS containing 0.3% Triton-100 for 20 min, and then sealed with 10% donkey serum for 30 min with PBS washes between each step. Next, the cells were incubated with anti-TLR2 (0.6 mg/mL diluted by 1:100), anti-KLK5 (1.295 mg/mL diluted by 1:100), and Alexa Fluor 488 (1:300) or 555 (1:300; Thermo Fisher Scientific) antibodies as primary and red- and green-fluorescently labeled secondary antibodies, respectively. Finally, DAPI was counterstained for 15 min. Images were captured using a Zeiss Axio Scope A1 (Zeiss, Oberkochen, Germany).

The study was conducted in accordance with relevant national legislation on the use of animals for research and the protocol was approved by the Ethics Committee of Sichuan province and Southwest Jiaotong University (Project identification code: SYXK(Chuan)2014-189). This experiment was carried out by implanting the bare 316L SS wires (Φ 0.1 mm × 10 mm) and PTMC-E5 coated 316L SS wires in the lumen of SD rats’ abdominal aorta for 1 month [31 (link)]. In brief, the wire remained in contact with flowing blood within the aorta to simulate the presence of a stent strut, with some regions of the wire in direct contact with the arterial wall and some not in contact. After 1 month, the aortas containing the implanted wires were harvested for histological analysis. Rat aortas containing the 316L SS wire implants were snap-frozen in liquid nitrogen and cryo-sectioned for histological analysis. Cross sections were ethanol-fixed and then stained with antibodies specific for EC (CD31, Sigma, Ontario, CA, USA), Smooth muscle cells (SMC, α-SMA, Sigma, Ontario, CA, USA) and macrophages (TNF-α, Sigma, Ontario, CA, USA). The nuclei of all the cells were stained by 4,6-diamino-2-phenyl indole (DAPI, Sigma, Ontario, CA, USA). The stained images were observed by confocal laser-scanning microscopy (CLSM, Nikon Eclipse Ti, Nikon, Tokyo, Japan).

ENS single cells were fixated with 4% formaldehyde in PBS at 5°C for 17 min on glas coverslips. Adult ENS networks were centrifuged, washed with PBS and separated from PBS residues. They were embedded in Tissue-Tek by freezing at −80°C. Tissue-Tek slices (10 μM) were made with a Kryostat and fixated with ice cold Acetone 70% (Applichem, 70% + 30% water). As primary antibodies rabbit anti iNOS (polyclonal, ab15323, Abcam), rabbit anti GFAP (polyclonal, Z0334, Dako, Germany) and mouse anti Tubulin β III (monoclonal, MAB1637, Merck Millipore, Germany) were used. Alexa Fluor 594 goat anti rabbit (Invitrogen, USA) and goat anti mouse 488 (Invitrogen, USA) were used as secondary antibodies. Rabbit IgG (Dako, Germany) was used for the isotype control. 4′,6-diamino-2-phenylindole (DAPI, Sigma-Aldrich, Germany) was used for nuclear stainings.