Whole brains were snap frozen in Tissue-Tek O.C.T. compound at 12 and 22 days after implantation (Sakura Finetex, Torrence, CA) fixed in 95% ethanol, rinsed in water, stained with Mayer's hematoxylin solution and Eosin Y solution (Sigma Aldrich, St. Louis, MO), dehydrated and mounted with Richard-Allen Scientific™ Mounting Medium (ThermoFisher, Waltham, MA). Immunofluorescent staining was performed on sections fixed in cold methanol for 10 min at -20°C, rinsed in PBS and incubated with primary antibodies, diluted in PBS containing 2% BSA, 5% goat serum, and 0.25% Triton X-100, overnight at 4°C. Antibodies for RABV nucleoprotein, NeuN, and CD4 have been described previously (31 (link)) and additional reagents are listed in Table 2. Slides were then incubated with fluorescence-conjugated secondary antibodies and mounted with Vectashield® Hard Set™ mounting medium (Vector Laboratories, Inc., Burlingame, CA) containing DAPI. Brightfield and fluorescent images were acquired with a Leica DM6000 microscope with the Leica Application Suite v4 program (Leica Microsystems, Switzerland). Image brightness and contrast were adjusted using Photoshop CS5 software.