Application suite v4 program

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.

Immunohistochemistry was performed as previously described (Lebrun et al. 2015 ). Briefly, brain tissues were embedded in OCT compound (Sakura Fintex, Torrance, CA) and blocks were cut using a Thermo Shandon cryostat (Pittsburgh, PA) into 15 µM sections. Sections were fixed and washed. After blocking, sections were incubated overnight in primary antibody (1:500) (Table I) at 4^⁰C. Secondary antibody (1:1000) was added for 2 hours at RT. Images were acquired with an upright Leica DM6000 microscope with the Leica Application Suite v4 program (Leica Microsystems, Heerbrugg, Switzerland). Brightness and contrast on pictures were adjusted using the GNU Image Manipulation Program.