Anti-MDM2 antibody [2A10] (ab16895) at 2 µg/ml + A549 whole cell lysate

Predicted band size: 55 kDa

Detection: chemiluminescence.

This image was generated using the ascites version of the product.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of ameloblastoma tissue sections labeling MDM2 with ab16895. The sections were de-paraffinized, hydrated and then rinsed in phosphate-buffered solution (PBS). They were immersed in heat-induced epitope retrieval citrate buffer of concentration 15mMol and pH 6.0, diluted 1/10 with distilled water and incubated at 95ºC for 10 minutes. They were then placed in fresh citrate, cooled in water for 20 minutes and then rinsed in PBS for 6 minutes. Peroxidase blocking reagent was added to each section for 5 minutes, and the sections were rinsed in 0.1% PBS for 6 minutes. The specimen were incubated for 30 minutes with 1/100 dilution of Anti-MDM2 antibody [2A10] (ab16895), then rinsed with PBS, followed by incubation with undiluted HRP for 20 minutes. 1ml of diaminobenzidine solution was added to cover the specimen, followed by incubation in a humidity chamber for 15 minutes. The sections were then immersed in aqueous haematoxylin and rinsed in distilled water for 5 minutes. The tissue was dehydrated and subsequently rinsed with xylene. Distyrene plasticizer in xylene mounting fluid was then applied, and a cover slip placed. Hematoxylin and eosin staining.

This image was generated using the ascites version of the product.

Overlay histogram showing HeLa cells stained with ab16895 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum (ab7481) / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab16895, 0.5µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This image was generated using the ascites version of the product.

ab16895 staining MDM2 in MCF7 cells treated with progesterone (ab141252), by ICC/IF. Decrease in MDM2 expression correlates with increased concentration of progesterone, as described in literature.
The cells were incubated at 37°C for 24 hour in media containing different concentrations of ab141252 (progesterone) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab16895 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

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Anti-MDM2 antibody [2A10] (ab16895) at 1/1000 dilution + Mouse Liver lysate at 40 µg with Milk, 2 hours at 25°C at 5 %

Secondary
Donkey anti-mouse IgG (HRP) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 55 kDa
Additional bands at: 55 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute

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ab16895 staining Human normal tonsil. Staining is localised to nuclear + cytoplasmic compartments. Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.

Sections were stained using an automated system DAKO Autostainer Plus, at RT: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 min. They were then blocked with Dako Protein block for 10 min (containing casein 0.25% in PBS) , incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 min. Colorimetric detection was completed with DAB for 5 min. Slides were counterstained with Haematoxylin and coverslipped under DePeX.

This image was generated using the ascites version of the product.

ab16895 (1/200) staining MDM2 in assynchronous HeLa cells (green). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). Please refer to Abreview for further experimental details.

This image was generated using the ascites version of the product.

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