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

Blocking and dilution buffer: 5% NFDM/TBST.

The expression of Neuropilin 1 and the mesothelial marker cytokeratin was analyzed in human peritoneal specimens by immunohistochemistry. Positive cells for antibodies used (Neuropilin 1 (ab81321) and Cytokeratin) show brown staining. Nuclei are counterstained in blue. (a, b) Control peritoneal tissue, with a conserved mesothelial cell monolayer showing an epithelioid morphology (with a 20X objective). These cells show weak expression of Neuropilin 1 and a marked staining for cytokeratin (arrows). No expression of these proteins was observed in the submesothelial area (region under mesothelial monolayer) (c, d) Fibrotic tissue sample from peritoneal dialysis (PD) patient showing the loss of mesothelial monolayer and invading spindle-like mesothelial cells in submesothelial area (with a 40X objective). These cells present a strong staining for Neuropilin 1 (c), and are also positive for cytokeratin (d) (arrows). Pictures are representative of 5 cases of PD patient samples and 4 of control samples.

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.

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.

Unpurified ab81321 staining Neuropilin 1 in the COS1 fibroblast-like cell line derived from monkey kidney tissue by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 0.1% and blocked with 10% serum for 60 minutes at 24°C. Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. An Alexa Fluor^® 488-conjugated goat anti-rabbit monoclonal(1/500) was used as the secondary antibody.

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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.

Blocking and dilution buffer: 5% NFDM/TBST.

Unpurified ab81321 staining Neuropilin 1 in rat brain tissue sections by Immunohistochemistry (frozen sections). Tissue was fixed with paraformaldehyde and then blocked with 10% serum for 1 hour at 27°C followed by incubation with the primary antibody, undiluted, for 14 hours at 4°C. An undiluted Cy3^® conjugated donkey anti-rabbit was used as the secondary antibody.

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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).

Unpurified ab81321 staining Neuropilin 1 in mouse brain tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% serum for 1 hour at room temperature; antigen retrieval was by heat mediation in citrate buffer (pH 6). Samples were incubated with primary antibody (1/100 in PBS + 2% blocking serum) for 16 hours at 4°C. A biotin-conjugated goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

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Immunohistochemical analysis of frozen human kidney tissue sections labelling Neurophilin 1 with ab81321 at a concentration of 1/100 for 18 hours at 4°C. The antibody was then blocked with a serum free protein block for 1 hour at 21°C. The secondary antibody used was a donkey anti-rabbit antibody conjugated to an Alexa488^® dye inclubated at 1/400.

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