Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreatic carcinoma tissue sections labeling Sonic Hedgehog  with purified ab53281 at 1/2000 dilution (0.097 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).

Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Sonic Hedgehog with purified ab53281 at 1/20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - cells without incubation with primary antibody and secondary antibody (Blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Sonic Hedgehog with purified  ab53281 at 1/250 dilution (0.8μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1/1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling Sonic Hedgehog with unpurified ab53281 at 1/20 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling Sonic Hedgehog with purified ab53281 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised by 0.1% tritonX-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Sonic Hedgehog with unpurified ab53281.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).

Immunohistochemical analysis of Formalin fixed paraffin-embedded human kidney cancerous tissue labeling Sonic Hedgehog with unpurified ab53281 at 1/100 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).

Immunohistochemical analysis of Formalin fixed paraffin embedded human ovarian cancer tissue, staining Sonic Hedgehog with unpurified ab53281.

Antigen retrieval was carried out in citrate buffer using a pressure cooker for 40 minutes. Sections were blocked with blocking agent before incubating with primary antibody (1/2000) for 90 minutes at room temperature. Staining was detected using DAB.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).

Overlay histogram showing HepG2 cells stained with unpurified ab53281 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab53281, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53281).