Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

This data was developed using ab184783 the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human kidney labelling Neuropilin 1 with ab81321 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab81321 Anti-Neuropilin 1 antibody [EPR3113] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control. Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

Clone EPR3113 (ab184783) has been successfully conjugated by Abcam. This image was generated using Anti-Neuropilin 1 antibody [EPR3113] (Alexa Fluor® 488). Please refer to ab197644 for protocol details.

ab197644 staining Neuropilin 1 in MCF7 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab197644 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

Clone EPR3113 (ab184783) has been successfully conjugated by Abcam. This image was generated using Anti-Neuropilin 1 antibody [EPR3113] (PE). Please refer to ab209445 for protocol details.

ab209445 staining Neuropilin 1 in MCF7 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab209445 at 1/100 dilution (Pseudocolored in green) and ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab81321).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

Clone EPR3113 (ab184783) has been successfully conjugated by Abcam. This image was generated using Anti-Neuropilin 1 antibody [EPR3113] (Alexa Fluor® 647). Please refer to ab198323 for protocol details.

ab198323 staining Neuropilin 1 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised in 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 ab198323 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 1/167 dilution overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab81321).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab81321).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab81321).

• IHC-P

AbReview19276****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab81321).

This image is courtesy of an Abreview submitted by Manoj Kumar Valluru.

• IP

Lab

Immunoprecipitation - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab81321).

All lanes:

Immunoprecipitation - Anti-Neuropilin 1 antibody [EPR3113] (<a href='/en-us/products/primary-antibodies/neuropilin-1-antibody-epr3113-ab81321'>ab81321</a>)

Predicted band size: 103 kDa

Observed band size: 120 kDa

false

Exposure time: 4s

• WB

Lab

Western blot - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

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.

All lanes:

Western blot - Anti-Neuropilin 1 antibody [EPR3113] (<a href='/en-us/products/primary-antibodies/neuropilin-1-antibody-epr3113-ab81321'>ab81321</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

NRP1 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human NRP1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-nrp1-knockout-a549-cell-line-ab269507'>ab269507</a>)

Lane 3:

MDA-MB-231 cell lysate at 20 µg

Lane 4:

SK-BR-3 cell lysate at 20 µg

Predicted band size: 103 kDa

Observed band size: 125-135 kDa

false

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (AB184783)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab81321).

Image from Pйrez-Lozano ML et al. PLoS One. 2013;8(4):e60776. Fig 5.; doi: 10.1371/journal.pone.0060776.