Newly eclosed male wCS flies were sorted in groups of 30 into vials with standard cornmeal medium. Vials were closed with two overlapping 3”×3”polypropylene mesh squares and tied with cotton twine. Vials were used within 6 hours. Four vials were placed in a 1000ml Nalgene straight sided jar with a 10ml glass beaker with 10ml of diacetyl 10−2 in paraffin oil. Jars were closed for exposure periods ranging from 2–6 days. In recovery experiments, vials were removed from the jars and were placed in a 25°C incubator for the remainder of the recovery period. Flies were anesthetized on ice, sacrificed in ethanol, and then immediately put into 1X phosphate buffered saline with 0.3% triton-x (PTX). Brains or antenna were put in 4% paraformaldehyde in PTX and incubated for 30min while rotating at 25°C. Samples were washed 5 times in PTX, and blocked in 5% natural goat serum in PTX for 1 hr while rotating at 25°C. Samples were then incubated in primary antibody with 5% goat serum in PTX, mouse-nc82 (1:20) (Development Studies Hybridoma Bank, University of Iowa) and rabbit-antiGFP (1:150) (Invitrogen) in PTX for 48 hours in 4°C while rotating. After washing 5 times in PTX samples were incubated in secondary antibody with 5% goat serum in PTX, rabbit-anti-Alexa488 (1:400) (Invitrogen) and mouse-antiAlexa568 (1:400) (Invirtogen) for 48 hours in 4°C. Samples are washed 5 times in PTX and stored in 70% glycerol in PTX. Images were taken using a Ziess 510 laser scanning confocal microscope, and image analysis was done using Image J software.
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