Chronic hIVIG treatment produces cell type specific changes in microglia. In order to assess whether chronic hIVIG treatment had differential effects on morphologically discernible phenotypes of microglia, we analyzed expression of Iba-1 and CD45 on each individual microglial cell along its entire X-Y-Z planes. Coronal sections of 35 μm were double stained with Iba-1 and CD45 primary antibodies followed by attaching fluorescent secondary antibodies. We then acquired confocal Z-stack images from hippocampi of both saline and hIVIG treated mice. After identifying and segregating the X-Y-Z boundaries of each microglial cell, a simple Image J macro measured the area, volume, and integrated densities for Iba-1 and CD45 markers across its Z-axis. Circularity was measured using maximum intensity projection image for each of the microglial stack. We analyzed 955 randomly chosen microglial cells spread across saline (n = 6) and hIVIG (n = 6) treated groups. (A) A 3-D scatter plot of CD45 and Iba-1 integrated intensities, and circularity for each microglia cell type. Green circles represent the hIVIG group and blue circles the saline group. Both visually and statistically, one can easily discern three clusters of cells which we designated as type A (n = 134; 14%), type B (n = 386; 40.4%), and type C (n = 417; 43.6%). The right panels illustrate representative cells of each type. Round morphology and high expression of CD45 alone characterizes type A cells. Type B cells features include ramified morphology with comparable expression of both CD45 and Iba-1 markers, whereas type C cells exhibit a ramified morphology but express predominantly Iba-1 marker. Scale bar = 5 μm. (B) The number of CD45 positive round cells encountered in brain parenchyma (cortex and hippocampus) of APdE9 vs. wild-type mice at 2, 4, 10, 15, and 19 months of age (n = 2, at each age point). APdE9 mice exhibit a much more conspicuous age-dependent increase in the number of CD45 positive round cells than their wild-type littermates.