HeLa cells were labelled with anti-ACA and anti-CENPF (ab5). ab5 was used at a working dilution of 1/400. This image demonstrates the dramatic increase in fluorescence that occurs late in G2 cells (indicated by arrows). In the final panel DAPI is pseudo-coloured blue, while ACA and CENPF are coloured green and red respectively. 40x magnification.

ICC/IF image of ab5 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab5, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. 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).

Ab5 staining Human normal colon tissue. Staining is localised to nuclear compartment.
Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Interpahse and Prophase HeLa cells were labelled with anti-ACA and anti-CENPF (ab5). ab5 was used at a working dilution of 1/400. This image emphasizes the redistribution of CENPF from the nuclear matrix during late G2 following entry into the initial stages of mitosis (see the accompanying image). Distinct punctate CENPF patterns proximally located in relation to the centromeres can be seen. In the final panel DAPI is pseudo-coloured blue, while ACA and CENPF are green and red respectively. 100x magnification. 

All lanes : Anti-CENPF antibody (ab5) at 1/1500 dilution

Lane 1 : Mitotic HeLa Lysate
Lane 2 : Asynchronous HeLa Lysate
Lane 3 : 50gamma Mitotic HeLa Lysate
Lane 4 : 50gamma Asynchronous HeLa Lysate

Lysates/proteins at 25 µg per lane.

Predicted band size: 330 kDa
Observed band size: 310 kDa why is the actual band size different from the predicted?

Paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized A549 (human lung carcinoma cell line) cells stained for CENPF (red) using ab5 at 1/500 dilution in ICC/IF. ). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 555) at 1/500 dilution was used as the secondary antibody.

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