All lanes: Anti-Shh Antibody (N-terminal) at 1:2000 dilution Lane 1: F9 whole cell lysate Lane 2: mouse stomach lysates Lane 3: rat liver whole cell lysates Lane 4: NIH/3T3 lysates Lysates/proteins at 20ug per lane. Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10,000 dilution Predicted band size: 48kD Blocking/Dilution buffer: 5% NFDM/TBST. Lysates/proteins at 20ug per lane. Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution. Predicted band size: 84kD Blocking/Dilution buffer: 5% NFDM/TBST.

Staining (Mouse) Shh in mouse cerebellum sections by Immunohistochemistry (Immunohistochemistry (paraffin) - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 3% BSA for 0.5 hour at room temperature; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/25) for 1 hours at 37°C. A undiluted biotinylated goat polyvalent antibody was used as the secondary antibody.

Overlay histogram showing HT-29 cells stained with Shh (green line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 90% methanol for 10 min. The cells were then icubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the antibody (Shh, 1:25 dilution) for 60 min at 37ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit lgG (H+L) (1583138) at 1/400 dilution for 40 min at 37ºC. Isotype control antibody (blue line) was rabbit IgG1 (1ug/1x10^6 cells) used under the same conditions. Acquisition of >10,000 events was performed.