ab136649 staining Nucleolin in human breast cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 5% Triton X-100 in PBS and blocked with 5% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/1000) for 1 hour. An undiluted Alexa Fluor® 546-conjugated anti-mouse IgG polyclonal was used as the secondary antibody.

See Abreview

All lanes : Anti-Nucleolin antibody [364-5] (ab136649) at 1 µg/ml

Lane 1 : HepG2 whole cell lysate at 20 µg
Lane 2 : HDFn whole cell lysate at 25 µg
Lane 3 : SH-SY5Y whole cell lysate at 20 µg

Secondary
All lanes : Goat polyclonal to Mouse IgG - AP at 1/3000 dilution

Predicted band size: 76 kDa

Immunocytochemical analysis of HeLa cells (paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15 min)) labelling Nucleolin with ab136649 at 1µg/ml (red) and anti-GAPDH (ab110305) in green, as a counter stain.
Secondary: Alexa 594 goat anti-mouse IgG1 at a 1/1000 dilution.

Immunocytochemical analysis of HDFn cells (paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15 min)) labelling Nucleolin with ab136649 at 1µg/ml (red) and anti-GAPDH (ab110305) in green, as a counter stain. Secondary: Alexa 594 goat anti-mouse IgG1 at a 1/1000 dilution.

Detection of Nucleolin by Immunoprecipitation.
Nucleolin in HepG2 cell lysate (1 mg) was immunoprecipitated using 1 µg ab136649 antibody. The gel was stained with Coomassie brilliant blue G.

Flow Cytometric analysis of HeLa cells (fixed and permeabilized with methanol) labelling Nucleolin with ab136649 (blue) at 1µg/ml or an equal amount of an isotype control antibody (red).

IHC image of nucleolin staining in human liver HCC formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab136649, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.