Colocalization of active Caspase 3 and Thy1.2 identifies male-reactive thymocytes undergoing clonal deletion throughout the cortex. Thymus tissue sections from mice in Fig. 3 were analyzed by immunofluorescence for Thy1.2, active Caspase 3, and G8.8. (A) Images were overlayed and examined for colocalization between Thy1.2 and active Caspase 3. White lines are drawn around G8.8⁺ cells to identify the medulla and boxes identify colocalized cells. C, cortex; M, medulla. Bar, 250 μm. (B) Colocalization was confirmed at high magnification. Bar, 5 μm. (C) The frequency of colocalization is expressed as the number of colocalized cells per total Thy1.2⁺ cells in ten different images per thymus. Data represent the mean from four different males ± SD. (D) The relative distance of colocalized cells from the medulla was digitally calculated using Photoshop. The drawn line indicates the median of the dataset. The entire figure is representative of two individual groups of chimeras and three replicates of immunofluorescence staining.