Secondary alexa fluor antibodies 488 and 546

Anti-eNOS (ab5589) and anti-iNOS (ab3523) used for immunohistochemistry were from Abcam (Paris, France). Anti-eNOS antibody (AF950) used for immunoprecipitation experiments was from R&D Systems (Bio-Techne, France). Anti-glutathione antibody recognizing GS-S-proteins was from Virogen (Watertown, MA, USA). Secondary antibodies anti-mouse and anti-rabbit HRP-conjugated were from Cell Signaling Technology (Ozyme, France). Anti-Von Willebrand Factor (VWF) (AB7356) was from Chemicon (Merck Millipore) and anti-VEGF was from Sigma. Secondary anti-goat HRP-conjugated was purchased from Southern Biotech (Clinisciences, France). Secondary Alexa Fluor antibodies (488 and 546) were from Life Technologies (Courtaboeuf, France). Dihydroethidine (DHE), DAF-FM diacetate (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate), dithiotreitol (DTT), 4,6-Diamidino 2-phenylindole dihydrochloride (DAPI), oxypurinol, VAS2870, L-NAME (N-Nitro-L-arginine methyl ester hydrochloride), BH4 (tetrahydrobiopterin dihydrochloride) were from Sigma-Aldrich (Saint Quentin Fallavier, France). 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) and SYTO-13 were from Thermofisher (Villebon sur Yvette, France), NOC-18 (diethylenetriamine/nitric oxide adduct; DETA NONOate), was from Santa Cruz Biotechnology (Clinisciences, France).

Cell culture reagents were from Invitrogen Life Technologies (Thermofisher). Anti-HNE-Michael adduct antibodies were from Oxis Research (#24327) for immunofluorescence studies, and from Invitrogen (#MA5-27570), for immunoprecipitation experiments. The anti-vimentin monoclonal antibody was from Abcam (#ab92547). Anti-γH2AX (#9718S), anti-SIRT1 (#9475S), anti-acetylated-Lysine (#9441S), and secondary anti-mouse and anti-rabbit HRP-conjugated antibodies were from Cell Signaling Technology. Anti-ubiquitin antibody was from Santa Cruz Biotechnology (#sc-8017). 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), L-Carnosine, 2-phenylindole dihydrochloride (DAPI), and anti β-actin antibody were from Sigma-Aldrich. Secondary Alexa Fluor antibodies 488 and 546 were from Life Technologies.

The fresh frozen sections were fixed in cold methanol (−20 °C for 20 min), washed with PBS several times and incubated with blocking solution (PBS supplemented with 1% BSA and 4% donkey serum) followed by the addition anti-CD36 (EMD Millipore, #MAB1258) and anti-acetyl-α-tubulin (Lys40) (Cell Signaling Technologie^®, # 5335S) antibodies. Subsequently, the sections were washed and primary antibody detection was conducted with compatible Alexa Fluor^® -488 and -546 secondary antibodies (Molecular Probes^®). Conventional immunofluorescence in fixed tissue was performed by incubating washed sections in blocking solution (PBS containing 4% donkey serum, 1% BSA, and 0.4% Triton™ X-100) for 30 min. Using the same buffer solution composition, the sections were incubated over night at 4 °C with primary antibody anti-OMP (WAKO, Ltd. Japan #544–10001) or anti-TUBB3 (Aviva System Biology, CA #ARP63784). After incubation with the primary antibody, OE sections were rinsed in PBS and incubated with compatible Alexa Fluor^® -488 and -546 secondary antibodies for 120 min. Sections were then rinsed with PBS and counterstained with DAPI for nuclear labeling. Staining controls were performed to assure the specificity of CD36 labeling (Fig. S3).