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).

HeLa cells stained with mouse monoclonal to Fibrillarin (green) and with chicken antibody to vimentin (red) and counterstained with a fluorescent DNA probe (blue). Nuclear DNA is revealed with DAPI(blue). The vimentin antibody was used at a dilution of 1/1000 and the fibrillarin monoclonal at 1/100. Cultures were processed using standard fixation and staining procedure (in protocol section).

All lanes : Anti-Fibrillarin antibody [38F3] - Nucleolar Marker (ab4566) at 1/500 dilution

Lane 2 : C6 cytosol fraction
Lane 3 : C6 nuclear fraction
Lane 4 : HEK-293 cytosol fraction
Lane 5 : HEK-293 nuclear fraction
Lane 6 : NIH-3T3 cytosol fraction
Lane 7 : NIH-3T3 nuclear fraction

Observed band size: 37 kDa why is the actual band size different from the predicted?

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).

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.

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

Lane 1 : cytoplasmic protein fraction of HeLa cells with LI-COR® Odyssey® Blocking Buffer, 45 minutes at room temperature at 50 %
Lane 2 : nuclear protein fraction of HeLa cells with LI-COR® Odyssey® Blocking Buffer, 45 minutes at room temperature at 50 %

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : AlexaFluor 680 goat anti-mouse at 1/10000 dilution

Performed under reducing conditions.

Additional bands at: 34 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 second

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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 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.

ab staining Fibrillarin in Human melanoma A7 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/500 in 1% BSA) for 24 hours at 4°C. A FITC-conjugated Goat anti-mouse polyclonal (1/200) was used as the secondary antibody.

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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).