This ImageJ macro was developed to perform automated quantitative analysis of microscopy images for the measurement of AgRP (Agouti-related peptide) and ATG5 (autophagy-related protein 5) signals in hypothalamic ARC (arcuate nucleus) regions. The macro processes multi-channel fluorescence images, extracts specific regions of interest (ROI), and calculates mean and max intensity values for AgRP and ATG5 signals. Key Features: Batch Processing: Processes all files with a specified suffix (.lif by default) in a given input directory and its subdirectories. Channel Selection: Allows user-defined selection of channels for AgRP, ATG5, and nuclei staining. Automated Segmentation: Uses Gaussian blur and Otsu thresholding to segment nuclei and calculate particle-based measurements. Data Export: Generates a summary CSV file containing image names, AgRP mean, AgRP max, and ATG5 mean values. Overlay Generation: Creates PNG overlays showing the segmentation results overlaid on the original channels.