Anti-NG2 antibody

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NG2 antibody (AB129051)

IHC image of NG2 staining in Human skin melanoma formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution

1. for 20 mins. The section was then incubated with ab129051, 1μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NG2 antibody (AB129051)

ab129051 staining NG2 in Malme-3M cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 ab129051 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

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Western blot - Anti-NG2 antibody (AB129051)

Abcam recommends using milk as the blocking agent - 3%. This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab129051 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

All lanes:

Western blot - Anti-NG2 antibody (ab129051) at 1 µg/mL

Lane 1:

Brain (Mouse) Tissue Lysate at 25 µg

Lane 2:

Mouse Hippocampus Tissue Lysate at 25 µg

Lane 3:

Cerebellum Mouse Tissue Lysate at 25 µg

Lane 4:

Spinal Cord (Mouse) Tissue Lysate at 25 µg

Lane 5:

Brain (Rat) Tissue Lysate at 25 µg

Lane 6:

Spinal Cord (Rat) Tissue Lysate at 25 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 251 kDa

Observed band size: 252 kDa

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Exposure time: 20min