Recombinant Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

False colour image of Western blot: Anti-Neuropilin 1 antibody [EPR3113] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab81321 was shown to bind specifically to Neuropilin 1. A band was observed at 125/135 kDa in wild-type A549 cell lysates with no signal observed at this size in NRP1 knockout cell line ab269507 (knockout cell lysate ab269669). To generate this image, wild-type and NRP1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Neuropilin 1 with purified ab81321 at 1/400. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

The expression of Neuropilin 1, VEGFR-2, and VEGF was analyzed by immunofluorescence microscopy in omentum and effluent-derived mesothelial cells (MCs). MCs were double stained for Neuropilin 1 (green) and VEGFR-2 (red), and single stained for VEGF (green). Nuclei were stained with DAPI. Neuropilin 1 and VEGF show a membrane distribution in omentum and epithelioid MCs (b, c, h, i). During in vitro (e, f) and ex vivo (k, l) MMT both proteins change their localization and are internalized. The expression of VEGFR-2 is down-regulated but it does not show differences in localization during in vitro (a, d) and ex vivo (g, j) MMT.

Blocking and dilution buffer: 5% NFDM/TBST.

Intracellular Flow Cytometry analysis of MCF7 cells labelling Neuropilin 1 with purified ab81321 at 1/70 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

Neuropilin 1 was immunoprecipitated from 0.35mg mouse heart lysate with ab81321 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab81321 at 1/1000 dilution (0.77 μg/mL). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/1000 dilution.

Lane 1: Mouse heart tissue lysate 10 μg
Lane 2: Mouse heart tissue lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab81321 in mouse heart lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Blocking and dilution buffer: 5% NFDM/TBST.

ICC/IF image of unpurified ab81321 stained MCF7 cells. The cells were 4% PFA 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 (unpurified ab81321, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) 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.

Overlay histogram showing HepG2 cells stained with unpurified ab81321 (red line). The cells were fixed with 4% paraformaldehyde (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 ab81321, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (0.5µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a significantly decreased signal in HepG2 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions. 

Blocking and dilution buffer: 5% NFDM/TBST.

Immunocytochemistry/Immunofluorescence analysis of HUVEC cells labelling Neuropilin 1 with purified ab81321 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).