Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: GPC3 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HepG2 whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab95363 observed at 70 kDa. Red - loading control, ab130007, observed at 125kDa.

ab95363 was shown to specifically react with Glypican 3 in wild-type HAP1 cells as signal was lost in GPC3 knockout cells. Wild-type and GPC3 knockout samples were subjected to SDS-PAGE. ab95363 and ab130007 (Mouse anti-vinculin loading control) were incubated overnight at 4°C at 1/1000 and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human hepatocellular carcinoma tissue sections labeling Glypican 3 with ab95363 at 1:100 dilution (4.66 ?g/ml). Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on human hepatocellular carcinoma, performed on a Leica Biosystems BOND^TM RX instrument.
The section was incubated with ab95363 for 30 mins at room temperature.

Immunocytochemistry/ Immunofluorescence analysis of HepG2 (human hepatocellular carcinoma epithelial cell) cells labeling Glypican 3 with purified ab95363 at 1/200 (2.3 µg/ml). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow cytometry analysis of HepG2 (human hepatocellular carcinoma) labeling Glypican 3 with purified ab95363 at 1/80 dilution (5.825 µg/ml) (red). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlableled control -Unlabelled cells (blue).

Immunohistochemical staining of human liver hepatocellular carcinoma with ab95363.

Flow cytometric analysis of rabbit anti-Glypican 3 (SP86) antibody ab98363 (1/100) in HEPG2 cellls (green) compared to negative control of rabbit IgG (blue).

ICC/IF image of ab95363 stained HepG2 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 ab95363 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.