Fv100 spectral confocal system

At either 0 h or 96 h postinfection, the medium was removed from EV-D68 virus-infected or uninfected organotypic brain slice cultures and the cultures were placed overnight in 4% paraformaldehyde (PFA) fixative dissolved in 1× PBS at 4°C. Following fixation, cultures were incubated in blocking solution of PBS, 0.3% Triton X-100, and 3% horse serum. Cultures were incubated overnight in at 4°C in blocking solution containing appropriate primary antibodies. Sections were washed in 1× PBS and incubated in the presence of fluorophore-conjugated secondary antibody in blocking solution. Sections were mounted on slides using Aqua-Poly/Mount (Polysciences, Inc.) or Fluorsave (Millipore, Fisher) and imaged using an iZ80 laser scanning confocal microscope (Olympus FV100 spectral confocal system). Brain sections were imaged using an 60× 1.42-numerical-aperture (NA) oil objective or a 10× 0.40-NA air objective. All images were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

LMMP tissues were fixed in Zamboni's fixative (4% paraformaldehyde and 0.2% piciric acid in 0.1 M PBS; #1459, Newcomer Supply, Middleton, WI) overnight at 4°C. The next day, tissues were rinsed in PBS until clear. Tissues were transferred to glass slides and incubated in blocking buffer [10% normal donkey serum (NDS), 0.5% Triton-X 100 in PBS] for 2 h at room temperature. Tissues were then incubated in appropriate primary antisera overnight at 4°C against green fluorescent protein (GFP; 1:500; ab13970, Abcam, Cambridge, MA), HuD (1:25; A-21271, Life Technologies, Eugene, OR), S100 Protein Ab-2 (S100; 1:200; RB-044-A0, Thermo Scientific, UK), choline acetyltransferase (ChAT; 1:50; AB144P, Millipore, Temecula, CA); vasoactive intestinal peptide (VIP; 1:200; Abcam), neuronal nitric oxide synthase (nNOS; 1:200; ab1376, Abcam), calretinin (1:200; CG1, Swant, Switzerland), or calbindin D-28K (1:200; AB1778, Millipore). The next day, tissues were rinsed 4 times for 10 min in PBS, then incubated in appropriate Alexa Fluor secondary antibodies at 1:200 for 2 h at room temperature. Antibody dilutent was 3% NDS, 0.5% Triton-X 100 in PBS. Tissues were then rinsed and slides were coverslipped with 2.5% PVA/DABCO. Fluorescent images were captured on an Olympus FV 100 Spectral Confocal system (Melville, NY) in the Ohio State University Campus Microscopy and Imaging Facility.