Anti pecam 1 m 20 antibody

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies (Tokyo, Japan). EPA, DHA, and 4-HHE were purchased from Cayman (Ann Arbor, MI, USA). Anti-β-actin antibody (A5316) and N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fatty acid-free bovine serum albumin (BSA) was purchased from Nacalai Tesque (Kyoto, Japan). Anti-epoxide hydrolase 2 (sEH) antibody was purchased from Gene Tex Inc. (Irvine, CA, USA). Anti-p38 kinase (p38) (#9212) and anti-phospho-p38 (#9211) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-PECAM-1 (M-20) antibody and anti-heme oxygenase 1 (HO-1) antibody were purchased from Santa Cruz (Dallas, TX, USA). Anti-cytochrome P450 2J2 (CYP2J2) antibody was purchased from Origene (Rockville, MD, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies were purchased from Amersham Biosciences Corp. (Piscataway, NJ, USA). Small interfering RNA (siRNA) was purchased from Life Technologies (Tokyo, Japan). SB203580 was purchased from Calbiochem (Cambridge, UK). All other reagents and chemicals were from standard suppliers.

Sections (4 μm thick) of formalin-fixed/paraffin-embedded human tumours excised from mice were used for immunohistochemical analysis. After de-paraffinization in xylene and rehydration in graded ethanol, antigen retrieval was performed by heating the sections in citrate buffer (10 mM, pH 6.0) for 5 min. Non-specific staining was blocked with a mixture of 10% goat serum and casein (2 × ) for 30 min at room temperature. The slides were incubated in rabbit polyclonal anti-PECAM-1 (M-20) antibody (Santa Cruz) (1:100 diluted in PBS containing 1 × casein) for 1.5 h at room temperature, followed by Texas Red conjugated goat anti-rabbit IgG antibody (Invitrogen) at 1:200 in PBS for 30 min at room temperature. Rabbit IgG was used as a negative control. Finally, the slides were mounted in Vectashield mounting media containing DAPI (Vector Laboratories, Burlingame, CA, USA).