Anti-Pericentrin antibody - Centrosome Marker (ab4448)

Human coronary artery endothelial cells (HCAEC) were labeled for Pericentrin using ab4448 at 1/100 dilution in ICC/IF. Primary incubation was for 1 hour at room temperature (green). The seciondary antibody was a Goat anti-rabbit IgG AlexaFluor^® conjugate.

Fixation: Formaldehyde. Permeabilisation: 0.1% Trition X-100 in HBSS.

Image courtesy of Human Protein Atlas

Paraffin embedded human kidney tissue sections were incubated with ab4448 (1/4500 dilution) for 30 minutes at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab4448 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further images can be found at www.proteinatlas.org

A representative panel of indirect immunofluorescence microscopic images shows normal (regular, n ≤ 2) and aberrant centrosome numbers (n > 2) in interphase cells.

Centrosomes were stained using anti-pericentrin antibody ab4448 (magenta), nuclear DNA is shown in blue (DAPI).

Statistical methods: Kruskal-Wallis test. Mann-Whitney U tests followed by Bonferroni-Holm p-value correction were made as post-hoc tests in order to compare the MDS and sAML patients with the control group.

IF staining of pericentrin in MCF7 (Human breast adenocarcinoma cell line) cells.

The top panel is an interphase cell showing centrosome staining. 

The bottom panel shows a mitotic cell with spindle pole staining. 

ab4448 was used at 1/500, but also works at higher dilutions (1/1000-1/2000).

Top panel - 630X magnification; Bottom panel -1000X magnification.

The secondary antibody was Alexa-Fluor^®488 anti-rabbit.

NIH/3T3 (Mouse embryo fibroblast cell line) cells were fixed in 100% methanol for 6 minutes at -20°C, washed 3 times in PBS then incubated with ab4448 (1/2000) for 1 hour at room temperature.

The panel of images shows the nuclei stained with DAPI (blue), ab4448 staining is shown in green. 100x magnification.

ICC/IF image of ab4448 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in methanol (5 minutes) and incubated with the antibody (ab4448, 1 µg/ml) for 1 hour at room temperature. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor^® 594 WGA was used to label plasma membranes (red).

ab4448 stained NIH/3T3 (Mouse embryo fibroblast cell line) cells.

The cells were fixed in 100% methanol for 5 minutes at -20°C and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab4448 at 5 µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1 hour at room temperature. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1 hour at room temperature.

DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1 hour at room temperature.

ICC/IF image of ab4448 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 100% methanol (5 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab4448, 1 µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1 hour. Alexa Fluor^® 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 1.43µM.