Anti-Glypican 3 antibody [SP86]

• Flow Cyt

Lab

Flow Cytometry - Anti-Glypican 3 antibody [SP86] (AB95363)

Flow cytometry overlay histogram showing left wild-type Hap1 positive cells and right negative GPC3 knockout Hap1 stained with ab95363 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab95363) (1x 106 in 100μl at 5.0 μg/ml (1/396)) for 30min on ice.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (black line) was used at the same concentration and conditions as the primary antibody. Unlabelled sample was also used as a control (blue line).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glypican 3 antibody [SP86] (AB95363)

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 μg/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™ RX instrument.
The section was incubated with ab95363 for 30 mins at room temperature.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Glypican 3 antibody [SP86] (AB95363)

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.

• Flow Cyt

Unknown

Flow Cytometry - Anti-Glypican 3 antibody [SP86] (AB95363)

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).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Glypican 3 antibody [SP86] (AB95363)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glypican 3 antibody [SP86] (AB95363)

ab95363, at 1/100 dilution, staining Glypican 3 in formalin-fixed, paraffin-embedded Human liver cancer tissue by Immunohistochemistry.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glypican 3 antibody [SP86] (AB95363)

Immunohistochemical staining of human liver hepatocellular carcinoma with ab95363.

• Flow Cyt

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Flow Cytometry - Anti-Glypican 3 antibody [SP86] (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).

• WB

Supplier Data

Western blot - Anti-Glypican 3 antibody [SP86] (AB95363)

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.

All lanes:

Western blot - Anti-Glypican 3 antibody [SP86] (ab95363)

Predicted band size: 66 kDa

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