Anti-ROR2 antibody [Nt 2535-2835] (ab190145)

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: ROR2 knockout HAP1 whole cell lysate (20 µg)
Lane 3: MCF7 whole cell lysate (20 µg)
Lane 4: U20S whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab190145 observed at 125 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab190145 was shown to recognize ROR2 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when ROR2 knockout samples were examined. Wild-type and ROR2 knockout samples were subjected to SDS-PAGE. Ab190145 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) ab216777 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

IHC image of ROR2 staining in Normal Human Stomach formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F/B. 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 ab190145, 10µg/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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ab190145 stained U20S cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab190145 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

IHC image of ROR2 staining in Human normal prostate 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 ab190145, 10µg/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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre