Anti mouse α sma antibody

The paraffin section was stained with hematoxylin and eosin (HE), Masson’s trichrome (MT) and Picrosirius red (PSR) [17 (link),23 (link)]. In immunohistochemical analysis, GFP antibody (Abcam plc, Tokyo, Japan), anti-mouse αSMA antibody (Abcam plc, Tokyo, Japan), anti-mouse CD31 antibody (Dianova GmbH, Hamburg, Germany), anti-mouse neutrophil antibody (NIME-R14: Abcam plc, Tokyo, Japan) and anti-mouse TGF-β1 antibody (Abcam plc, Tokyo, Japan). An EnVision Kit (DAKO Japan Inc., Tokyo, Japan) was used for visualization. Semi-quantitative evaluation was performed: negative (Grade 0), slightly positive (Grade 0.5), positive (Grade 1) and strongly positive (Grade 2).

With the standard protocol being followed prior to HE staining described above, the sections were subjected to antigen retrieval and blocking as previously described [15 (link)]. For the immunohistochemical analysis, the sections were initially incubated with mouse anti-α-SMA antibody (Abcam, Cambridge, MA, USA) or mouse anti-TGF-β1 antibody (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 4°C overnight and were then incubated with HRP-conjugated goat antimouse secondary antibody (Abcam, Cambridge, MA, USA) at 37°C for 30 min. Positive staining was detected with HRP-conjugated streptavidin, visualized with 3,3′-diaminobenzidine, and counterstained with hematoxylin. Finally, the sections were mounted and cover-slipped, and images of five representative fields at ×400 magnification were captured by the Leica QWin Plus v3 software (Leica Microsystems, Cambridge, UK). The sizes of the study areas and the integrated optical density (IOD) of the positive stains were measured using Image-Pro Plus 5.1 (Media Cybernetics, Rockville, MD, USA). The expression levels of the proteins were expressed as the mean optical density (MOD; MOD = IOD per unit of study area).

Plasmid purification kits (QIAGEN Plasmid Maxi Kit, QIAprep Spin Miniprep Kit) were purchased from QIAGEN (Venlo, The Netherlands). Restriction enzyme, T4 polynucleotide kinase, alkaline phosphatase (E. coli C75), DNA ligation kit (DNA Ligation Kit Ver.1), DNA polymerase (TAKARA Premix Taq, EX Taq version), and Site-Directed Mutagenesis kit (Mutan^®- Super Express Km) were purchased from Takara Bio (Kusatsu, Japan). Heparin was purchased from Mochida Pharmaceuticals (Shinjuku City, Tokyo) and Block Ace from Sumitomo Dainippon Pharma (Osaka, Japan). Blue Sepharose 6-Fast Flow, 5 mL HiTrap Phenyl HP, and 5 mL HiTrap Q XL were purchased from GE Healthcare Japan (Tokyo, Japan). HE staining reagents were purchased from Muto Chemical (Tokyo, Japan). Mouse anti-α-SMA antibody (cat#:ab5694) was purchased from abcam (Cambridge, UK). All other reagents and solvents were commercially available special grade products, and the water used as the solvent was ion-exchanged water or Milli-Q water.

Vascular tissue slides were perforated by 0.1% triton (X100, Sigma) for 15 minutes and then incubated with blocking solution (10% normal goat serum in PBS diluent) for 1 hour. The slides were then washed with PBS for 5 minutes, and slices were incubated with mouse anti‐α‐SMA antibody (1:500; Abcam), ICAM‐1 (1:500; Abcam) or VCAM‐1(1:500; Abcam) mixed with rabbit anti‐FoxO1 antibody (1:100; Cell Signaling Technology) at 4 °C overnight. Primary antibodies were then poured away and washed with PBS 3 times, 10 minutes for each wash. Secondary antibodies (1:1000, Goat anti‐mouse Alexa‐568 and Goat anti‐rabbit EGFP‐488; Cell Signaling Technology). were added and incubated for 1 hour followed by washing with PBS again 3 times, for 10 minutes each time. DAPI (D9542, Sigma) was added for 20 minutes, followed by another 3 times of PBS washing, each time for 5 minutes. Slides were then mounted with mountant (06 522, Sigma), covered with coverslip, dried overnight and checked with a microscope connected with computer using ZEN 2012 (Version 2.3).

Collagenase type II, Elastase, and Exisulind were obtained from Sigma-Aldrich co. (St Louis, MO, USA). PDGF-BB was purchased from R&D systems (Minneapolis, MN, USA). Goat anti-PKG I, rabbit anti-VE-cadherin, mouse anti-osteoponin, rabbit anti-CD31, and goat anti-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-VASP (Ser239), rabbit anti-t-Akt, and mouse anti-p-Akt (Ser473) were purchased from Cell Signaling (Berkely, MA, USA). Mouse anti-calponin antibody was obtained from Sigma-Aldrich co. (St Louis, MO, USA). Mouse anti-α-SMA antibody was purchased from Abcam (Cambridge, MA, USA). The secondary antibodies to each primary antibody were as follows. Anti-mouse IgG HRP conjugated and anti-rabbit IgG HRP conjugated antibodies were obtained from Promega (Madison, WI, USA). Anti-goat IgG HRP conjugated antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse IgG Alexa flour 488 and anti-rabbit IgG Alexa flour 594 secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Alzet osmotic pump was purchased from Durect corporation (Cupertino, CA, USA).