Ez c1

Two million cells were plated on a chamber slide followed by infection with HCMV and treatment with emetine or GCV for 24 or 72 h. Cells were fixed with 3.7% paraformaldehyde for 20 min at room temperature, permeabilized with ice cold methanol for 10 min at -20°C and blocked with 5% bovine serum albumin in 0.5% Tween-20 for 20 min at room temperature. Cells were incubated with primary antibodies at 4°C overnight, washed and incubated with fluorescently-labeled secondary antibodies for 2 h at 37°C. Fluorescence microscopy was performed using a confocal laser scanning microscope (Nikon EZ C1). All images were captured at 60X magnification and processed under identical conditions with constant parameters (including scan speed and excitation and emission wavelengths) using Nikon EZ C1 software. Data analysis (percent nuclear localization) was performed by NIS-Elements software (Nikon) for a minimum of 40 cells in the high-density and 25 cells in the low-density samples from two fields per condition.

Images were captured using a Nikon C1 Plus microscope (from Nikon in Tokyo, Japan), equipped with the Nikon C1 confocal system and four lasers (403 nm, 561 nm, 643 nm, and Argon tunable 458/477/488/515 nm), and operated via Nikon’s EZ-C1 software. The 435–465-nm filter was also used to detect autofluorescence. Each optical section of each channel series was scanned twice using Nikon EZ-C1 Average. For all images shown, a series of optical sections was collected, and a subset of this series was used to project the images using either Nikon EZ-C1’s Volume Render, Maximum function or ImageJ’s 3D Projection, Max function. ImageJ (from the National Institutes of Health in Bethesda, MD, United States) was used to open projected images, separate and combine color channels, and adjust contrast and brightness of images.

Brains of different groups of 30-day-old flies were dissected in cold PBS and fixed in 4% paraformaldehyde in PBS for 1 h. The samples were washed and permeabilized overnight in 0.3% Triton X-100 in PBS (Wash buffer). Fixed brains were stained with mouse anti-TH antibody (mouse monoclonal, Immunostar)/anti-α-syn antibody (Syn1- Rabbit polyclonal, BD Biosciences)/anti-α-syn filament antibody (rabbit monoclonal, cat number ab209538; Abcam) at a 1:200 dilution for 48 h. After incubation, the brains were washed three times and incubated with the corresponding secondary antibodies overnight (anti-mouse Alexa 488/anti-rabbit Alexa 594) at 1:1000 dilution. After thorough washing, the stained brains were mounted using Fluoromount mounting medium (Sigma), and images were acquired using a confocal microscope (Nikon EZC1). DA neuron clusters were analyzed as previously described [38 (link)]. Optical sections of the brains were acquired at 40-μm intervals using a 40× objective for whole-brain imaging. Confocal stacks were merged into a single plane using Nikon EZC1 software (Nikon, Tokyo, Japan). The number of TH-positive neurons within the PPL1 and PPL2 DA neuronal clusters was counted by visual inspection of the individual confocal Z-series of images. An average of eight brains were analyzed in both hemispheres, for each genotype, and the results are expressed as the mean ± S.E.M.