Axiovert spinning disk confocal microscope

Using two pairs of fine tweezers, brains were dissected from adult flies in PBS. Within a 15 minute span, brains were collected into a wire mesh container soaked in PBS and then were fixed for 20 minutes in PBS containing 4% paraformaldehyde. Sindbis-GFP infected fly brains were washed three times in PBS and then mounted onto slides using Prolong Gold antifade reagent if no other additional staining was necessary (Life Technologies). Otherwise, SINV-GFP dissections were treated the same as VSV-infected brains. Dissected, VSV infected brains were washed three times in PBS. Brains were then blocked in blocking buffer (PBS containing 0.5% bovine serum albumin and 0.2% Triton X-100) for 30 minutes. After three washes in wash buffer (PBS containing 0.2% Triton X-100), brains were stained with primary antibodies overnight at 4°C. After three 10-minute washes, brains were incubated in Alexafluor secondary antibodies (Life Technologies) diluted to 1:600 for 1.5 hours at room temperature. DRAQ5 stain (ThermoFisher 62251) was added for an additional 30-minute incubation. Brains were washed three more times at 10 minute intervals before being mounted to glass slides using Prolong Gold antifade reagent. Imaging was done on a conventional fluorescent microscope or a Zeiss axiovert spinning disk confocal microscope, and ImageJ software was used for image analysis.