IHC image of YAP1 staining in human colon adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab39361, 1µg/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-YAP1 antibody (ab39361) at 1 µg/mlLane 1 : SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate with 5% BSA blockingLane 2 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate with 5% BSA blockingLane 3 : LS147T (Human colon cancer) Whole Cell Lysate with 5% BSA blockingLane 4 : SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate with 1% milk blockingLane 5 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate with 1% milk blockingLane 6 : LS147T (Human colon cancer) Whole Cell Lysate with 1% milk blockingLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP))developed using the ECL techniquePerformed under reducing conditions.
ICC/IF image of ab39361 stained HCT116 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab39361, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.