Elements advanced research software

To measure induction of the ldIs3 [gcs-1::gfp] oxidative stress response reporter, L4 LD1171 ldIs3 or JRP1024 fshr-1(ok778); ldIs3 worms were transferred to SK plates with PA14 or OP50 prepared as if for a pathogen killing assay. After 5 hours, worms were anesthetized with 25 mM NaN₃, then photographed using a Nikon 90i compound epiflourescence microscope. To quantify reporter expression, the intestine was manually outlined using the Nikon Elements Advanced Research software. The mean fluorescence intensity of this Region of Interest was determined by the software for at least 10 worms per condition.
To validate the microarray data, reporter strains BC11778 sEx11778 [gpx-1::gfp] and BC14266 sEx14266 [gst-38::gfp] [42 ] were transferred as L4 larvae to SK plates with PA14 or OP50 prepared as described above. After 5 hours, worms were anesthetized and photographed as described above. The exposure time was held constant for all conditions within an experiment.

Malpighian tubules were isolated from 0 to 6 h OrR adults and imaginal discs from 3rd-instar wandering larvae (a mixture of males and females) by manual dissection in Grace’s unsupplemented media. Tissues were fixed with 4% formaldehyde in PBS for 10 min, washed, and stained with 50 ng/mL DAPI for 10 min at room temperature. Tissues were mounted on slides and imaged on a Nikon Eclipse Ti epifluorescence microscope with a 60X oil-immersion objective using a Hamamatsu ORCA-ERA camera. DAPI intensities were measured for individual nuclei using the Nikon Elements Advanced Research software. DAPI intensities for adult Malpighian tubule nuclei were normalized to diploid larval imaginal disc nuclei to calculate ploidy.

Live T. brucei cells (5 × 10⁶) expressing TbTim17 mutants were used for MitoTracker staining as previously described (69 (link)). Briefly, cells were washed twice with PBS and spread evenly over gelatin-coated slides. Once the cells had settled, the slides were washed with cold PBS to remove any unattached cells. The attached cells were fixed with 3.7% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% non-fat milk for 30 min, the slides were washed with 1× PBS. DNA was stained with 1 µg/mL 4′,6-diamidino-2-phenylindole. Cells were imaged using a Nikon TE2000E widefield microscope equipped with a 60 × 1.4 NA Plan Apo VC oil immersion objective. Images were captured using a CoolSNAP HQ2 cooled CCD camera and Nikon Elements Advanced Research Software. There were no max projections of a Z-stack. The software used to calculate the Pearson’s coefficient is Nikon NIS Elements Advanced Research Imaging Software.

Brain sections (35 µm) were maintained at −20 °C in cryoprotectant, stained while free-floating, and mounted to glass slides for imaging, using a “primary antibody delete” (secondary antibody only) stained section to establish background fluorescence limits. Fluorescent immunohistochemical images were collected using an Olympus BX61 confocal microscope and Fluoview 1000 software (Melville, NY). Quantitative fluorescence measurements were thoroughly monitored using standard operating imaging parameters to ensure that images contained no saturated pixels. For quantitative comparisons, all imaging parameters (e.g., laser power, exposure, and pinhole) were held constant across specimens. Confocal images were analyzed using Nikon NIS-Elements Advanced Research software (Version 4.5, Nikon, Melville, NY). A minimum of six images per tissue slice were analyzed per animal, averaging 9–15 neurons per 60–100× image (approximately 180 cells per animal, per histological stain). 20× magnification was used to generate montage imaging of the ventral midbrain, for which the entire SN was analyzed per image using anatomical region of interest (ROI) boundaries. Results are reported as a measure of puncta within TH-positive cells, either number of objects (# of objects), or area per neuron in square pixels (px²), generated by Nikon Elements Advanced Research software.