Lipid droplet quantification with ImageJ

To give a consistent approximation of adrenal size in sections, the left adrenals were sectioned through their centers and along the longest axis. Sections with a cut plane at the center showing a clear and large medulla area were used for quantification. Confocal pictures (10 from each section with no overlaps) were taken from the middle zone of the adrenal cortex (zona fasciculata). LD and nuclei signals from the same image were analyzed separately using ImageJ v1.52 software (NIH) to quantify the number of nuclei and the size and numbers of LDs. Settings for signal enhancements and thresholds were adjusted for a representative picture, and identical settings were used for all images from the same experiment. To validate the quantification procedure, 10 different images were taken from each of three adrenal sections from the same adrenal, and the procedure was repeated in four mice. Experimental variance was found to be lower between sections than between individual images from the same section. Based on these results, we concluded that a high number of images taken from one carefully picked representative section per animal gave the best quantitative data.