Anti-VEGF antibody [VG-1] (ab1316)

Immunocytochemistry/ Immunofluorescence - VEGF antibody [VG1] (ab1316)

ab1316 at 10µg/ml staining human retinal pigment epithelial cells. The antibody was incubated with the cells for 6 hours and then detected with Alexa-Fluor ® 568 goat anti-mouse (IgG) antibody.

This image is courtesy of an Abreview submitted on 16 November 2005.

Western blot - VEGF antibody [VG-1] (ab1316)

All lanes : Anti-VEGF antibody [VG-1] (ab1316) at 10 µg/ml

Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/3000 dilution

Predicted band size : 24, 45 kDa
Observed band size : 43 kDa (why is the actual band size different from the predicted?)
Additional bands at : 125 kDa,52 kDa. We are unsure as to the identity of these extra bands.

Immunocytochemistry/ Immunofluorescence - VEGF antibody [VG-1] (ab1316)

ICC/IF image of ab1316 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab1316, 10µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 10µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 10µg/ml.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VEGF antibody [VG-1] (ab1316)

ab1316 staining VEGF in Human MDA-MD-231 cells injected into the mouse mammary fat pad by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde, permeabilized with Tween in PBS and blocked with 1.5% serum for 1 hour at 25ºC; antigen retrieval was by heat mediation in citrate buffer. Tissue samples were incubated with primary antibody (1/200 in PBST +1% BSA) for 16 hours at 4ºC. A biotin-conjugated Goat anti-mouse IgG polyclonal (1/200) was used as the secondary antibody. Tissue was counterstained with Hematoxylin (1/10) for 30 seconds at room temperature and rinsed with water.

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VEGF antibody [VG-1] (ab1316)

IHC image of ab1316 staining in human heart formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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 ab1316, 5µg/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.