Image analysis for in situ hybridization

Autoradiographic film images of hybridized brain sections were captured by a digital camera with four sections sampled/animal and averaged to provide a single mean. Semiquantitative analyses of autoradiograph images were performed using Scion Image software, and hybridization signal was expressed as corrected gray level. The gray-level signal of the region of interest was corrected by subtracting the background gray-level signal. Standards were also measured using Scion Image to verify that all quantified gray levels were on the linear range of the film.