Plasma Shh Quantification by ELISA

Plasma levels of Shh were assessed through ELISA assay as previously described (21 (link), 26 (link)). Briefly, 50 µl of each plasma sample was loaded in duplicate into the 96-well Shh ELISA plate (ab100639, Abcam Cambridge, UK). After 2.5 h of incubation at room temperature (RT), wells were washed and probed for 1 h at RT with Biotinylated Shh detection antibody. Then, wells were washed and probed with HRP-Streptavidin for 45 min at RT. Wells were washed and the revelation was performed by adding in each well 100 µl of a TMB substrate following by the addition of a Stop solution. Optical densities at 450 nm were determined with a plate reader, the Multiscan GO reader, V.1.01.10 (ThermoFisher Scientific, France). The concentration of Shh in each sample was determined using standard controls (recombinant proteins) and the generated standard curve.