Visualizing Midgut RNAi in Western Corn Rootworm

Fifty diet-raised 2^nd instar WCR were treated and surface-sterilized as previously described [15 (link)], with modifications (S1 Method). Twenty midguts were dissected and rinsed in sterile 1x PBS to eliminate gut contents. Pretreated midguts were incubated with Cy3-labeled dvssj1 dsRNA and siRNAs at 10 ng/μl in insect medium (EX-CELL 420 Serum-Free Medium; Sigma) for 15 hours at 25°C protected from light. Unconjugated Cy3 dye was used as a control at the same concentration. Midguts were washed twice with 1x PBS and fixed in 4% paraformaldehyde for 1 hour at room temp. Then midguts were washed three times with 1x PBST (PBS +0.1% Tween) for 5 minutes each. Samples were counterstained with DAPI (Sigma, 10 μg/μl of stock diluted to 1:1000) for 5 min and washed once in 1x PBST. Individual guts were placed on glass slides with 2x SlowFade concentrated Slow Fade antifade reagent in PBS, then coverslipped and imaged on a Leica TCS SPE. The 405 nm laser line was used for excitation of DAPI staining and the 532 nm laser line was used for excitation of Cy3 staining. Image analyses were carried out as described in S1 Method.