Recombinant Anti-VEGFA antibody [EP1176Y] - C-terminal (ab52917)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling VEGFA with purified ab52917 at 1:100 dilution (2.96 µg/ml).

Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0.

Negative control shown in inset.

Purified ab52917 staining VEGF in NIH/3T3 (Mouse embryo fibroblast cell line) cells.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with purified ab52917 at 5 μg/ml and ab195884, at 1/250 dilution, overnight at +4°C, followed by a further incubation at room temperature for 1h with an Goat anti-Rabbit Alexa Fluor^®488 secondary (ab150081) at 2 μg/ml (shown in green).

Nuclear DNA was labeled in blue with DAPI.

Overlay histogram showing NIH/3T3 (Mouse embryo fibroblast cell line) cells stained withunpurified ab52917 (red line). 

The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab52917, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 0.1 µg/1x10⁶ cells used under the same conditions. 

Unlabeled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. 

This antibody gave a positive signal in NIH/3T3 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used at 1/100 dilution. 

Unpurified ab52917 staining VEGF in mouse kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 5% serum for 45 minutes at 25°C. Samples were incubated with primary antibody (1/400 in 4% BSA + 5% serum in PBST) for 14 hours at 4°C. An Alexa Fluor^® 546-conjugated Donkey anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

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Intracellular Flow Cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling VEGFA with purified ab52917 at 1/30 dilution (10µg/ml) (red). 

Cells were fixed with 4% paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluorr^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). 

Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).