ab9669 at 2.5 µg/ml staining human breast invasive ductal carcinoma tissue by IHC-P.  The anitbody was incubated for two hours at room temperature. An HRP-labeled polymer detection system was used with a DAB chromogen. Heat induced antigen retrieval with a pH 6.0 Sodium Citrate buffer is recommended. 

developed using the ECL techniquePerformed under reducing conditions.HL-60 cells were incubated at 37°C for 24hrs with vehicle control (0 µM) and varied concentrations of 2-Arachidonylglycerol (ab120098). Increased expression of MCP1 in HL-60 cells correlates with an increase in 2-Arachidonylglycerol concentration, as described in literature.Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab9669 at 1 µg/ml and ab8227 at 1 µg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

Anti-MCP1 antibody (ab9669) at 1/4000 dilution + lysate prepared from human smooth muscle treated with 100µg/ml glycated LDL at 10 µgSecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/20000 dilutiondeveloped using the ECL techniqueObserved band size : 12-14 kDa (why is the actual band size different from the predicted?)Exposure time : 1 minuteImage courtesy of Dr Laura Toma by Abreview.See Abreview