Fak primary antibody

The slides were deparaffinized, followed by antigen retrieval. Endogenous peroxide and nonspecific proteins were blocked. The slides were then incubated with a FAK primary antibody (#05-537, Millipore, Billerica, MA) in a blocking solution overnight at 4°C. After washing, the horseradish peroxidase-conjugated secondary antibody was added. The slides were stained with DAB substrate and counterstained with hematoxylin. The expression of FAK was interpreted and graded. Scores of 0 or 1+ were considered negative and those of 2+ or 3+ were considered moderately or strongly positive for FAK overexpression.

The immunohistochemical staining was performed with FAK 4.47 antibody (Millipore #05-537). For antigen retrieval, slides were heated in the microwave for 10 minutes in citrate buffer (pH 6.0), followed by a 15 minute cool period. Endogenous peroxidase was quenched with aqueous 3% H₂O₂ for 10 minutes and washed with 1×PBS with 0.5% Tween 20 solution. Slides were loaded on a DAKO autostainer and blocked with serum-free protein block solution (Dako #X0909) which was applied for 5 minutes and then FAK primary antibody (Millipore #05-537) was applied for one hour. The biotinylated goat anti-mouse IgG (Jackson Immuno Research Labs, #115-065-062) was applied for 30 minutes, followed by the Elite ABC Kit (Vectastain, #PK-6200) for 30 minutes, and the DAB chromagen (Dako, #K4007) for 5 minutes. The slides were counterstained with hematoxylin, dehydrated, cleared and cover slipped.