Western blot - Neuropilin 1 antibody [EPR3113] (ab81321)

All lanes : Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/1000 dilution

Lane 1 : Human placenta lysate
Lane 2 : HUVEC cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : mouse heart tissue lysate
Lane 5 : mouse kidney tissue lysate
Lane 6 : rat heart tissue lysate
Lane 7 : rat kidney tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
HRP goat anti rabbit at 1/2000 dilution

Predicted band size : 103 kDa
Observed band size : 120 kDa (why is the actual band size different from the predicted?)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Neuropilin 1 antibody [EPR3113] (ab81321)

Immunohistochemical analysis of paraffin-embedded human kidney tissue using ab81321 at 1/100 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Neuropilin 1 antibody [EPR3113] (ab81321)

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 4ºC. A biotin-conjugated Goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

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

Immunohistochemistry (Frozen sections) - Neuropilin 1 antibody [EPR3113] (ab81321)

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.

Image courtesy of an anonymous Abreview.

Flow Cytometry-Neuropilin 1 antibody [EPR3113](ab81321)

Overlay histogram showing HepG2 cells stained with 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 (ab81321, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. 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.

Immunocytochemistry/ Immunofluorescence - Neuropilin 1 antibody [EPR3113] (ab81321)

ICC/IF image of 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 (ab81321, 5µg/ml) overnight at +4ºC. 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.