Immunocytochemistry/ Immunofluorescence - Fibrillarin antibody [38F3] - Nucleolar Marker (ab4566)

Rat neurons and glial stained with mouse monoclonal to Fibrillarin (green) and with chicken antibody to neurofilament NF-H (red). Cells were counterstained with a fluorescent DNA probe (blue). Nuclear DNA is revealed with Hoechst dye (blue). Cultures were processed using our standard fixation and staining procedure (in protocol section).

Immunocytochemistry/ Immunofluorescence - Fibrillarin antibody [38F3] - Nucleolar Marker (ab4566)

Human neuroblastoma line SH-SY5Y stained with mouse monoclonal to Fibrillarin (green) and with chicken antibody to neurofilament NF-H (red) and counterstained with a fluorescent DNA probe (blue). Nuclear DNA is revealed with Hoechst dye (blue). The NF-H antibody was used at a dilution of 1/100000 and the fibrillarin monoclonal at 1/1000. Cultures were processed using standard fixation and staining procedure (in protocol section).

Immunocytochemistry/ Immunofluorescence - Fibrillarin antibody [38F3] - Nucleolar Marker (ab4566)

High magnification view of human Hek293 cell nuclei stained with mouse monoclonal to fibrillarin (green), counterstained with a fluorescent DNA probe (blue). Nuclear DNA is revealed with Hoechst dye (blue). Cultures were processed using our standard fixation and staining procedure (in protocol section).

Western blot - Anti-Fibrillarin antibody [38F3] - Nucleolar Marker (ab4566)

All lanes : Anti-Fibrillarin antibody [38F3] - Nucleolar Marker (ab4566) at 1/2000 dilution (+ blocking buffer 1:1 in PBS + 0.1% Tween for 1 hour)

Lane 1 : Lysate of the cytoplasmic protein fraction of HeLa cells
Lane 2 : Lysate of the nuclear protein fraction of HeLa cells

Lysates/proteins at 20 µg per lane.

Secondary
An Alexa Fluor^®680-conjugated Goat anti-mouse polyclonal at 1/10000 dilution

Performed under reducing conditions.

Observed band size : 34 kDa (why is the actual band size different from the predicted?)


Blocking Step: 50% Li-COR^® Odyssey^® Bloc for 45 minutes at room temperature

Cell fractionation was performed as described in Woodhouse BC et al DNA Repair (Amst). 2008: 7(6):932-40.

This image is courtesy of an Abreview submitted by Svetlana Khoronenkova

Flow Cytometry-Fibrillarin antibody [38F3] - Nucleolar Marker(ab4566)

Overlay histogram showing HEK293 cells stained with ab4566 (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 / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab4566, 1/20 dilution) 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 IgG1 [ICIGG1](ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Western blot - Anti-Fibrillarin antibody [38F3] - Nucleolar Marker (ab4566)

Mouse embryonic fibroblast fractionation.

Cytopl - cytoplasmic fraction.
Nucl - nuclear fraction.
20 µg of each loaded.

ab4566 used at a 1/2000 dilution.
The secondary used was an Alexa-Fluor 680 conjugated goat anti-mouse polyclonal used at a 1/10000 dilution.

Image courtesy of an anonymous Abreview.