Recombinant Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

Purified ab52987 staining p75 NGF receptor in paraffin embedded Human tonsil tissue sections by Immunohistochemistry. Antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at 3.3μg/ml. A ready to use Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on germinal centre of human tonsil.

The differentiation capacity of purified CD34⁺ cells cultured for 3 days in the presence or absence of ALK5i was evaluated by performing immunofluorescence analysis assessing whether CD34⁺ cells had changed to cells expressing p75 and/or α-SMA. Expressions of CD34, p75 and α-SMA were assessed by immunofluorescence on day 0 (F-H), day 3 in SP+f medium (I-K), or day 3 in the same medium as (I-K) but with ALK5i (L-N).

Cultured re-aggregates were fixed in 4% PFA and embedded in paraffin. Sections (5 ∝m) were boiled in 0.01 M citrate (pH 6.0) with 0.1% Tween 20 for 10 min, washed three times in 0.1% Tween-20/PBS, transferred to blocking solution containing 5% BSA and 5% horse serum (Sigma) or goat serum (Invitrogen) in 0.1% Triton X-100/PBS for 1 hr, and incubated with primary antibody (p75 at 1/100 dilution) at 4°C overnight. After washing, the secondary antibody was added, and the sections were incubated for 2 hrs at room temperature. Microscopic images were obtained using a CCD camera (DP72, Olympus, Tokyo) mounted on a fluorescence microscope (BX60, or BX61VS-ASW, Olympus). Cultured cells on coverglasses were fixed in 4% PFA. Antigen retrieval was done by incubation with 100% methanol (-20°C) 10 min, and 0.3% Triton X-100 for 10 min.

Blocking and diluting buffer: 5% NFDM/TBST

ab52987 fails to detect band of interest in Capan-1 (positive, PMID: 14613990) and brain lysates (positive, PMID: 21413144, 21541365), indicating its low affinity in some p75 NGF Receptor positive materials.

Intracellular Flow Cytometry analysis of PC-12 (rat adrenal gland pheochromocytoma) cells labeling p75 NGF Receptor with purified ab52987 at 1/80 dilution (1ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control. 

Lane 1 (input): PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate 10μg
Lane 2 (+): PC-12 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52987 in PC-12 whole cell lysate

ab52987 immunoprecipitating p75 NGF receptor in PC-12 whole cell lysates. For western blotting, primary antibody used was ab52987 at 1.6 μg/ml. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution. Capture antibody was used at 1:40 dilution (2μg in 0.35mg lysates).

Blocking and diluting buffer: 5% NFDM/TBST.

Exposure: 10 seconds

Purified ab52987 staining p75 NGF receptor in PC-12 (rat adrenal gland pheochromocytoma) by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100. Samples were incubated with primary antibody at 3.9 µg/ml. An AlexaFluor^®488 Goat anti-Rabbit was used as the secondary antibody at 2 µg/ml. DAPI was used as a nuclear counterstain. Confocal image showing cytoplasmic and Membranous staining in PC-12 cells.

Blocking and diluting buffer: 5% NFDM/TBST

ab52987 fails to detect band of interest in hippocampus and cortex lysates (positive, PMID: 25180603, 28507518, 18930453, 20937383, 21059364), indicating its low affinity in some p75 NGF Receptor positive materials.

Exposure: Lane 1-3: 8 seconds
                 Lane 4-6: 180 seconds
                 Lane 7-9: 30 seconds

ICC/IF image of ab52987 stained PC12 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52987, 1 µg/mL) overnight at 4^oC. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluo^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.4 µM.

Intracellular Flow Cytometry analysis of PC-12 (rat adrenal gland pheochromocytoma) cells labeling p75 NGF Receptor with unpurified ab52987 at 1/60 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.