All lanes : Anti-S100A4 antibody [EPR2761(2)] (ab124805) at 1/1000 dilution

Lane 1 : Wild-type A549 whole cell lysate
Lane 2 : S100A4 knockout A549 whole cell lysate
Lane 3 : A375 whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 12 kDa
Observed band size: 12 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab124805 observed at 12 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab124805 was shown to recognize S100A4 in wild-type A549 cells as signal was lost at the expected MW in S100A4 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and S100A4 knockout samples were subjected to SDS-PAGE. Ab124805 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-S100A4 antibody [EPR2761(2)] (ab124805) at 1/1000 dilution (unpurified)

Lane 1 : Human tonsil lysate
Lane 2 : A549 cell lysate
Lane 3 : A375 cell lysate
Lane 4 : Human small intestine lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 12 kDa
Observed band size: 12 kDa

Unpurified ab124805 staining S100A4 in the A549 cell line from Human lungs by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 0.25% in PBS. Samples were incubated with primary antibody (1/100) for 45 minutes at 25°C. A TRITC-conjugated Goat anti-rabbit  polyclonal was used as the secondary antibody.

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ab124805 staining S100A4 in HeLa (human cervix adenocarcinoma) by flow cytometry. Cells were fixed with 80% methanol and permeabilised with 0.1% Triton X-100 (in PBS). The sample was incubated with the primary antibody at a dilution of 1/800. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: ab172730 rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

ab124805 staining S100A4 in HeLa (human cervix adenocarcinoma) by flow cytometry. Cells were fixed with 80% methanol and permeabilised with 0.1% Triton X-100 (in PBS). The sample was incubated with the primary antibody at a dilution of 1/80. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: ab172730 rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

ab124805 staining S100A4 in human tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab124805 immunoprecipitating S100A4. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/30 and Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at a dilution of 1/1500.

Lane 1: Human tonsil whole cell lysate (10ug)
Lane 2: Human tonsil whole cell lysate 
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab124805 in human tonsil whole cell lysate

Anti-S100A4 antibody [EPR2761(2)] (ab124805) + A549 (human lung carcinoma) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 12 kDa
Additional bands at: 12 kDa. We are unsure as to the identity of these extra bands.

Blocking buffer: 5% NFDM /TBST 

Diluting Buffer: 5% NFDM /TBST 

All lanes : Anti-S100A4 antibody [EPR2761(2)] (ab124805) at 1/2500 dilution

Lane 1 : Human tonsil tissue lysate
Lane 2 : A375 (human malignant melanoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 12 kDa
Additional bands at: 12 kDa. We are unsure as to the identity of these extra bands.

Blocking Buffer: 5% NFDM /TBST

Diluting Buffer: 5% NFDM /TBST 

Unpurified ab124805, at 1/250 dilution, staining S100A4 in paraffin-embedded Human tonsil tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical analysis of Human lung tissue, staining S100A4 with unpurified ab124805.

Tissue was fixed with HOPE and blocked with blocking solution for 5 minutes at 25°C. Samples were incubated with primary antibody (1/1000 in diluent) for 1 hour at 25°C. An undiluted HRP-conjugated goat anti-rabbit polyclonal IgG was used as the secondary antibody.

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ab124805 staining S100A4 in Jurkat (human acute T cell leukemia) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at 1/500. ab7291 and ab150120 were used as counterstains for primary antibody ab124805 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)