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1. Observation of SKN-1 Localization in Response to Streptococcus gordonii Infection
- Add ~100 L4 larvae to each Todd–Hewitt yeast extract (THY) seeded with Streptococcus and to each nematode growth medium (NGM) agar plate seeded with Escherichia coli (E. coli). Use three plates per strain of bacteria. Incubate the plates for 2 to 3 h at 25 °C.
- Thereafter, remove the plates from the incubator, wash them with a modified M9 buffer (M9W), and collect the worms in 15 mL conical tubes.
- Wash the worms 3x.
- Remove most of the M9W by aspiration and add 500 µL of M9W containing 2 mM sodium azide or 2 mM tetramisole hydrochloride to the worm pellet. This will anesthetize the worms, ensuring that no movement occurs when imaged under the microscope.
CAUTION: Use personal protective equipment (PPE) when handling sodium azide. Prepare the azide solution under a chemical hood. - Incubate the worm pellets at room temperature (RT) for 15 min. Then, spot 15 µL of the worm suspension onto a prepared agarose pad. Gently place a no. 1.5 coverslip over the agarose pad containing the anesthetized worms.
- Using a fluorescent microscope, visualize the localization of Skinhead family transcription factor 1 or SKN-1:GFP (Green Fluorescent Protein-tagged SKN-1) utilizing fluorescein isothiocyanate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI) filters. Image worms at 10x and 20x magnifications.
- Score the worms based on the level of localization of SKN-1. No nuclear localization, localization of SKN-1B/C::GFP (Green Fluorescent Protein-tagged SKN-1 isoform B/C) in the anterior or posterior of the worm, and nuclear localization of SKN-1B/C::GFP in all intestinal cells are categorized as low, medium, and high levels of localization, respectively.
- After scoring the fluorescent micrographs, determine the statistical differences by chi- squared and Fisher's exact tests using statistical software.