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Our Abpromise guarantee covers the use of ab2254 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 0.5 - 1 µg/ml.
Methanol fixation recommended.
|IP||Use at an assay dependent concentration.|
|WB||1/1000 - 1/2000. Detects a band of approximately 39 kDa (predicted molecular weight: 39 kDa).|
Immunofluorescence in human cells using Rabbit polyclonal to Aurora B (red), DAPI (blue) and CREST serum (binds to centromeres)(green).
(a) HeLa cells - transition from interphase (left) through mitosis
(b) RPE-1 cells - as in (a)
(c) HeLa cells - interphase
(d) RPE-1 cells - interphase
Blocked with 2% BSA.
IHC image of Aurora B staining in Human Lymph node Hodgkins diseaseformalin 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 ab2254, 5µg/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 image of ab2254 stained HeLa 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 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2254, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) 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.
ab2254 staining human A431 (epithelial) cells by ICC/IF. The sample was fixed in paraformaldehyde and permeabilized by incubation with 0.1% Triton X100. 1% BSA was used as the blocking agent prior to a 1 hour incubation with the primary antibody, diluted 1/1000 with 1% BSA made up in PBS. An Alexa Fluor® 647 conjugated Donkey anti-Rabbit IgG (H+L) antibody was used as the secondary. Blocking and antibody incubation steps were carried out at room temperature.
In this set of images, the tubulin is stained green, Aurora B in pink and DNA in blue.
Rabbit polyclonal to Aurora B (ab2254) used to stain SW620 human tumour xenografts (in mouse).
The sections were microwave pretreated in citrate buffer (pH 6.0) for 5 mins high then 5 mins simmer (800W conventional microwave). Slides were then incubated for 1 hour with the Aurora B primary antibody diluted 1/200 in TBS, then visualised using DAB, after application of an appropriate secondary.
The top panel shows paraformaldehyde fixed HeLa cells stained with ab2254 (1/1000) and counterstained with DAPI (red). Staining with ab2254 is shown in green. In the lower panel the staining with ab2254 is quenched by the addition of the blocking peptide, ab13569.
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