Anti-SATB2 antibody [EPNCIR130A] (ab92446)

Unpurified ab92446 staining SATB2 in mouse brain tissue by Immunohistochemistry (Frozen sections). Tissue was fixed with paraformaldehyde and permeabilized using 0.3% Triton-X-100. Samples were then blocked with 5% serum for 1 hour 30 minutes at 20°C followed by incubation with the primary antibody at a 1/200 dilution for 36 hours. A biotin conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/200 dilution.

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Lanes 1 - 2: Merged signal (red and green). Green - ab92446 observed at 83 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab92446 was shown to specifically react with SATB2 in wild-type HAP1 cells as signal was lost in SATB2 knockout cells. Wild-type and SATB2 knockout samples were subjected to SDS-PAGE. Ab92446 and ab9484 (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.

Unpurified ab92446 staining SATB2 in E18 Mouse brain tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde, permeablized with 0.3% Triton-X 100 and blocked with 3% BSA for 30 minutes at 25°C. The sample was incubated with primary antibody (1/500 in TBS with 0.1% Triton-X 100 + 3% Goat serum) at 4°C for 12 hours. An Alexa Fluor^® 546-conjugated Goat anti-rabbit polyclonal (1/1000) was used as the secondary antibody.

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Immunocytochemistry/Immunofluorescence analysis of SH-SY5Y (human neuroblastoma cell line from bone marrow) cells labelling SATB2 (green) with purified ab92446 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/100) and secondary antibody ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Flow Cytometry analysis of SH-SY5Y cells (human neuroblastoma cell line from bone marrow)  labeling SATB2 with purified ab92446 at 1/150 dilution(10ug/ml) (red). Cells were fixed with 4% paraformaldehyde  and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(ab150077)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black)(ab172730) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebral cortex tissue labelling SATB2 with purified ab92446 at 1/150. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.