Macro_quantificationISH

For ISH performed on embryos injected unilaterally, comparison of the expression levels between injected and control sides was assessed using a specific macro from ImageJ v2.1.0/1.53c (Abràmoff et al., 2004; Schneider et al., 2012). The macro functions based on the RGB color mode. RGB images are split into three channels (red, green, and blue) and pixel values corresponding only to the blue channel are recorded, excluding the red and green channels, since the signal recorded on the blue channel represents the expression levels. For each image, the region of interest (ROI) was specified, and its dimensions were fixed, such that the same ROI is placed on the control and injected side of the embryo which prevents any subjectivity in ROI determination. Measured are the area corresponding to the blue signal; the mean or average value of signal within the selected ROI; and the integrated density which is the equivalent of the product of area and mean, as it sums the values of pixels in the selection. In this study, ratio of integrated density measured in the injected versus control side was assessed. The macro ican be used as plug-in in ImageJ.