IHC image of CD146 staining in human aorta formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab24577, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

All lanes : Anti-CD146 antibody [P1H12] (ab24577) at 5 µg/ml

Lane 1 : HUVEC (Human Umbilical Vein Endothelial Cell) Whole Cell Lysate
Lane 2 : Human blood vessel: artery normal tissue lysate - membrane extract (ab28989)

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?
Additional bands at: 55 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes


This blot was produced using a 10% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab24577 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.