Axioplan fluorescent compound microscope

The protocol was modified from Kiyama et al., (2012) (link). Briefly, Fluoresbrite Plain YG 45.58um +/−0.80um solid latex microspheres in water (Polysciences Inc. 9003-53-6, catalog #18242, lot# 589756) were dissolved in 90% ethanol at a concentration of 3.64×10^10 and diluted 1:1000 in S-basal. 10^8 microspheres were applied to 60mm NGM dishes and spread with a flame-sterilized glass spreader. The S-basal was allowed to soak into the NGM overnight and protected from light. Worms were washed 3-4 times in a tabletop microcentrifuge with 1ml of S-basal + 0.1% Tween-20 (SBTw) to remove bacteria, and then applied to a dish. Dishes were incubated with open lids for 15 minutes in the dark, and then 1ml of 50mM sodium azide in SBTw was added to immobilize the worms. Worms were transferred to an Eppendorf tube and pelleted by centrifugation; the supernatant was removed, and ice-cold methanol was added for 10 minutes. Worms were washed once with SBTw and applied to large pre-made 1.5% agarose pads on glass slides with a rectangular coverslip. Slides were imaged on a Zeiss Axioplan fluorescent compound microscope equipped with a Zeiss AxioCam MRm digital camera using a 5x objective.

Slow killing was assayed as described (Estes et al., 2010 (link); Powell and Ausubel, 2008 (link)). Briefly, the P. aeruginosa PA14 strain was cultured in LB medium, seeded on 6 cm Petri dishes with 50 μM FUdR, incubated for 24 hours at 37°C and then for 24 hours at room temperature. 20-30 L4 stage hermaphrodites were transferred to these dishes, incubated at 25°C, and scored for survival every 12 hours. Experiments were repeated at least five times.
Two transgenic strains had pathogen responsive promoters driving GFP: F35E12.5 (acIs101[(F35E12.5::gfp);pRF4(rol-6(su1006))], and irg-1 (agIs17[irg-1::GFP; myo-2::mCherry]) (Estes et al., 2010 (link); Troemel et al., 2006 ). One transgenic strain had the phm-2 promoter driving GFP (amEx315[(phm-2p::gfp); pRF4(rol-6)]. To measure GFP induction, we placed 30–40 young adult hermaphrodites (one day past the L4 stage) on SK dishes with P. aeruginosa PA14 and cultured for 24 hours. Animals were washed 3 times with M9 and placed on a large pre-made 1.5% agarose pad with a 20ul drop of 1M sodium azide. A rectangular coverslip was applied, and slides were imaged on a Zeiss Axioplan fluorescent compound microscope equipped with a Zeiss AxioCam MRm digital camera using a 10x objective.