Immunocytochemistry/ Immunofluorescence - CD47 antibody [B6H12.2] (ab3283)

ab3283 staining CD47 in HUVEC cells by Immunocytochemistry/ Immunofluorescence.

The cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.25% Triton X-100 for 5 minutes and blocked with 1% BSA in 0.1% Tween 20 and PBS for 1 hour at room temperature. The cells were incubated in a mixture of two primary antibodies ab3283 and a rabbit anti-human VEGFR2 overnight at 4 ºC. The cells were then incubated with a mixture of two secondary antibodies (Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-mouse) in 1% BSA for 1 hour at room temperature in the dark. Counterstained with DAPI.

Image from Kaur S et al, J Biol Chem. 2010 Dec 10;285(50):38923-32. Epub 2010 Oct 5, Fig 5.

Immunocytochemistry/ Immunofluorescence - CD47 antibody [B6H12.2] (ab3283)

ab3283 at 1µg/ml staining CD47 from human OVCA3 cells by ICC. The cells were methanol fixed, blocked with 3% serum and incubated with the primary antibody for 30 minutes. A biotinylated horse anti-mouse IgG was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Mr Benjamin Misemer

Western blot - CD47 antibody [B6H12.2] (ab3283)

Anti-CD47 antibody [B6H12.2] (ab3283) at 1 µg/ml + Brain (Human) Tissue Lysate - adult normal tissue (ab29466) at 10 µg

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 35 kDa
Observed band size : 49 kDa (why is the actual band size different from the predicted?)


Exposure time : 4 minutes

CD47 contains an exstensive number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

Flow Cytometry - CD47 antibody [B6H12.2] (ab3283)

Overlay histogram showing peripheral blood lymphocytes stained with ab3283 (red line). The cells were incubated with the antibody (ab3283, 1µg/1x10⁶ cells) for 30 min at 4ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (ab96879) at 1/200 dilution for 30 min at 4ºC. 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 gating on peripheral blood lymphocytes.