Axio imager m2 stereoscopic microscope

Synchronized 65-hour-old animals were prepared using the method describe above. GFP-tagged bacteria were prepared using the method described above with the addition of selective antibiotics (S. enterica SL1344::gfp – 50 μg/mL of kanamycin; E. faecalis OG1RF::gfp – 100 μg/mL rifampicin) in the media plates. Synchronized animals were transferred to the plates seeded with GFP-tagged bacteria and incubated for 24 hours at 25°C. Following incubation, the animals were transferred to an empty NGM plate for 15 minutes to eliminate any fluorescent bacteria stuck to the body. The animals were then transferred to a new, empty NGM plate and into a droplet of M9 buffer to further wash away any bacteria remaining on the body. Animals were anesthetized using 25 mM Sodium Azide and placed on a 2% agarose pad. A drop of halocarbon oil was placed over the anesthetized animals to minimize bubbles and other artifacts before being covered with a glass coverslip. The animals were visualized using a Zeiss Axio Imager M2 stereoscopic microscope.

CL2070(dvls70) and nmur-1(ok1387);CL2070(dvls70) animals were synchronized as previously mentioned for RNA isolation. Synchronized L1 larval animals were transferred onto modified NGM plates seeded with E. coli HT115 and grown at 20°C for 48 hours until the animals had reached the L4 larval stage. The animals were collected and washed with M9 buffer before being transferred to NGM plates seeded with E. coli HT115 that were preheated to 35°C. Animals were then placed into a 35°C incubator for 2 hours. After the 2-hour incubation at 35°C, the animals were transferred to a 20°C incubator and allowed to recover for 2 hours. Animals were anesthetized using 25 mM Sodium Azide and placed on a fresh 2% agarose pad. A drop of halocarbon oil was placed over the anesthetized animals to minimize bubbles and other artifacts before being covered with a glass coverslip. The animals were visualized using a Zeiss Axio Imager M2 stereoscopic microscope. GFP quantification was performed using ImageJ, individual animal GFP expression were measured for the mean gray-value. An unoccupied section of the agarose pad was measured for its mean gray-value to account for background fluorescence.