Ab8488

For immunohistochemistry of aortic sinus sections endogenous peroxidases were blocked by immersion in 3% v/v hydrogen peroxide in PBS for 10 min. Antigen retrieval was performed with 10% v/v pH6 citrate buffer (Dako, Germany) in water at 95°C for 20 min and sections were then permeabilized with 0.5% v/v triton X-100 (Sigma) for 5 min at room temperature. Incubation in milk buffer for 30 min was used to block nonspecific antibody binding. Following washing in PBS, sections were incubated with primary antibodies to osteopontin (ab8488, Abcam, UK) at 1∶150 dilution overnight at 4°C, then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (P0448, Dako, Denmark) at 1∶200 dilution for 30 min. After repeated washing in PBS, complexes were visualized with diaminobenzidine and sections counterstained with Carazzi’s haematoxylin. Staining was quantified by image analysis software (NIS-elements Br 3.0, Nikon Instruments, USA).

Antibodies were directed to Ki-67 (AB16667, Abcam), calponin (SC136987, Santa Cruz), BECN1 (sc-11427, Santa Cruz), LC3B (#3868, Cell Signaling), LAMP1 (sc-17768, Santa Cruz), F4/80 (ab6640, Abcam), CD11b (PA5-18727, Invitrogen), Sca1 (ab-51317, Abcam), Osteopontin (ab8488, Abcam), LC3A/B (ab58610, Abcam). Secondary antibodies for immunofluorescence were Alexa Green 488 nm and Alexa Orange-Red 546 nm (Invitrogen). 4,6-diamidino-2-phenylindole (DAPI) (D3571), Prolong Gold Anti-fade mounting reagent (P36930), Slow-Fade Anti-fade reagent (S2828). Osteopontin was obtained from SIGMA (Cat. # O2260).