Anti-Aurora B antibody (ab2254)

ab2254 stained in Hela cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab2254 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

AURKB is expressed in mouse oocytes.

(Panel D) 20 GV-intact oocytes were collected from CF1 mice and micro-injected with the indicated cRNA. Two hours after injection, the oocytes were matured to Met II in vitro (16 h). The total numbers of non-injected control oocytes (Non-inj.) are indicated in parenthesis. Total cellular lysates were probed with the indicated antibody. The panels are images of the same membrane that was stripped and re-probed. The arrows indicate the specific AURKB protein band, and the asterisk indicates a presumed degradation product of AURKB-GFP.

AURKB is expressed in mouse oocytes.

(Panel A) GV-intact oocytes were collected from CF1 mice and matured in vitro for 8 h (Met I), or 16 h (Met II), prior to fixation and staining with an anti-AURKB antibody (ab2254).

HCT 116 (Human colorectal carcinoma cell line) cells were examined by immunofluorescence confocal microscopy.

Cells were treated with 500 nM AK301 for 16 hours, and then processed for Aurora B (ab2254) and β-tubulin staining (Panel B). The color key and 20 μm bars are shown. Images of representative field is shown with a 20 μm bar. End-labeled DNA is shown in red and DAPI-stained DNA is blue.

Cells cultured on coverslips were fixed with 4% paraformaldehyde at room temperature or 100% ice cold methanol at 4°C and then permeabilized with 0.5% Triton X-100 in PBS. Cells were blocked in 5% serum (in PBS) and then incubated with primary antibody (in 5% serum) on shaker for 1 h at room temperature.

Blocked with 2% BSA.

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

IHC image of Aurora B staining in Human Lymph node Hodgkins disease 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 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

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.

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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.