Irak phosphor t209

BCA sections were de-paraffinized and rehydrated in xylene and ethanol series. After antigen retrieval (H-3300, Vector Laboratories), sections were blocked with fish skin gelatin–PBS (6 g/L) containing 10% horse serum for 1 hour at room temperature. Slides were incubated with the following antibodies: mouse monoclonal SM α-actin-FITC (ACTA2) (4.4 μg/mL, clone 1A4, Sigma Aldrich), goat polyclonal anti-GFP (4 μg/mL, ab6673, Abcam) for detection of YFP, LGALS3 (2 μg/mL, Cedarlane CL8942AP), RUNX2 (1.374 μg/mL, ab192256, Abcam), Ki67 (4 μg/mL, ab15580, Abcam), PECAM-1 (1 μg/mL, Santa Cruz), IRAK phosphor-T209 (4 μg/mL, Abcam), iNOS (0.52 μg/mL, ab15323, Abcam), and Arg1 (9.2 μg/mL, GTX109242, GeneTex). The secondary antibodies were donkey anti-rat conjugated to Alexa 555 (5 μg/mL, Abcam), donkey anti-rat conjugated to Alexa 647 (5 μg/mL, Abcam), donkey anti-goat conjugated to Alexa 555 (5 μg/mL, Invitrogen), and donkey anti-goat conjugated to Alexa 647 (4 μg/mL, Invitrogen). Apoptosis was assessed by TUNEL (CF488A, Biotium) and cleaved caspase 3 staining (0.84 ug/mL, ab9661, Cell Signaling Technology). IL-1β antibody was detected using a donkey anti-mouse IgG2a-Alexa 488 antibody. DAPI (0.05 mg/mL, D3571, ThermoFisher Scientific) was used as a nuclear counterstain and slides were mounted using Prolong Gold Antifade (Invitrogen).