A method to quantify glial fibrillary acidic protein immunoreactivity on the suprachiasmatic nucleus

The aim of the present study was to develop a quantitative method to measure the immunoreactivity of the glial fibrillary acidic protein on the suprachiasmatic nucleus of hamster. For this purpose, optical microscopy images from brain sections processed for glial fibrillary acidic protein immunostaining were digitised under different light conditions. Image treatment and immunoreactivity quantification were performed following five different methods using the program Adobe Photoshop. The results were analysed in order to determine the ability of each method to differentiate immunoreactivity levels, and their susceptibility to the light conditions during image acquisition. Four of the five methods were found to be susceptible to the tested light conditions, while the other was not. This last method, which permits detection of differences in immunoreactivity between the different sections, was considered for further quantification of glial fibrillary acidic protein immunoreactivity on the suprachiasmatic nucleus.