ab10288 at a 1/500 dilution staining human dermal lymphatic endothelial by Immunocytochemistry. The cells were grown in coverslips and fixed in formalin prior to permeabilization with tritonX-100. The cells were incubated with the primary antibody at 1/500 for 1 hour and then bound antibody was detected using a biotinylated goat anti-mouse antibody. Texas red-streptavidin was used for development.

This image is courtesy of an Abreview submitted by Maria Navarro.

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All lanes : Anti-Podoplanin / gp36 antibody [18H5] (ab10288) at 5 µg/ml

Lane 1 : Human placenta tissue lysate - total protein (ab29745)
Lane 2 : Human skeletal muscle tissue lysate - total protein (ab29330)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 24 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 20 minutes


Podoplanin protein is extensively glycosylated. This might explain the observation of a band at a higher molecular weight than predicted.

Overlay histogram showing HCT116 cells stained with ab10288 (red line). The cells were fixed with methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab10288, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in HCT116 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.

Please note that Abcam does not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.

Western blot analysis of Podoplanin expression in human lymphatic endothelial cells (LEC) - lane 1, and HUVECs - lane 2. Total lysate of both cell types were subjected to SDS-PAGE and subsequent western blot analysis with ab10288. The antibody recognizes a protein of about 36 kDa in total lysate from LECs but not from HUVEC.

Immunocytochemistry/Immunofluorescence analysis of human dermal lymphatic endothelial cells (HDLEC) labelling Podoplanin / gp36 with ab10288. An Alexa Fluor^® 488-conjugated goat anti-mouse was used as the secondary antibody. Nuclei counterstained with DAPI.