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.

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.

Anti-Sonic Hedgehog antibody [EP1190Y] (ab53281) at 1/2000 dilution (purified) + Human fetal liver lysate at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 51 kDa
Observed band size: 27,50 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST.

50kDa: Full-length SHH; 27KDa: C-terminal product

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).

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).

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

Anti-Sonic Hedgehog antibody [EP1190Y] (ab53281) at 1/1000 dilution (purified) + Human fetal kidney lysate at 15 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 51 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM /TBST .

27KDa: C-terminal product.

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.

Anti-Sonic Hedgehog antibody [EP1190Y] (ab53281) at 1/2000 dilution (unpurified) + Fetal liver membrane at 10 µg

Secondary
Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa
Additional bands at: 27 kDa (possible isoform), 49 kDa. We are unsure as to the identity of these extra bands.

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

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.

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.