Anti-Fibrillarin antibody - Nucleolar Marker (ab5821)

  Immunocytochemistry/ Immunofluorescence - Fibrillarin antibody - Nucleolar Marker (ab5821)

ICC/IF image of ab5821 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab5821, 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).

  Immunofluorescence - Fibrillarin antibody - Nucleolar Marker (ab5821)

Immunofluorescent imaging of human cells (U2OS) with ab5821 reveals highly specific localisation to the dense fibrillar component (DFC) of the nucleolus associated with the initial ribosomal RNA (rRNA) precursor. The nucleolar protein fibrillarin is located primarily in the DFC.  Blue is hoechst staining of the nucleus, green is ab5821 used at 1/100, merge image demonstrates exclusively nuclear localisation.  

IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes.  All blocking and incubation steps carried out at 37 degrees C.

Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK

  Western blot - Fibrillarin antibody - Nucleolar Marker (ab5821)

All lanes : Anti-Fibrillarin antibody - Nucleolar Marker (ab5821) at 1 µg/ml

Lane 1 : Hela nuclear lysate
Lane 2 : Hela whole cell lysate
Lane 3 : A431 whole cell lysate
Lane 4 : Jurkat whole cell lysate
Lane 5 : 293T whole cell lysate
Lane 6 : Hela nuclear lysate with Fibrillarin peptide (ab13745) at 1 µg/ml
Lane 7 : Hela whole cell lysate with Fibrillarin peptide (ab13745) at 1 µg/ml
Lane 8 : A431 whole cell lysate with Fibrillarin peptide (ab13745) at 1 µg/ml
Lane 9 : Jurkat whole cell lysate with Fibrillarin peptide (ab13745) at 1 µg/ml
Lane 10 : 293T whole cell lysate with Fibrillarin peptide (ab13745) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 34 kDa


Exposure time : 10 seconds

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Fibrillarin antibody - Nucleolar Marker (ab5821)

ab5821 staining human liver tissue sections by IHC-P.  Sections were formaldehyde fixed, permeabilized in Triton-X and subjected to heat mediated antigen retrieval prior to blocking with 6% BSA for 2 hours at 23°C. The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 5°C.  ab6717 was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Dr Tom Donndelinger

See Abreview