Plan apochromat 20 0.8 m27 objective

Epifluorescence microscopy images were collected to confirm autofluorescence measurements. Preparation included culturing cells as a monolayer in 35 mm glass bottom micro well dishes (MatTek Corporation, Ashland, MA) following the previously mentioned growth protocol. Cells were washed with PBS before imaging. In order to capture endogenous fluorescence of NAD(P)H and morphological cell features, an Axio Observer Z1/7 scope with Plan‐Apochromat 20×/0.8 M27 objective was used (ZEISS, Oberkochen, Germany). Bright field images were obtained using the Axiocam 506‐3 V TL lamp. Autofluorescence of NAD(P)H was captured using a 335–383 nm excitation filter and 420–470 nm emission filter corresponding to the NAD(P)H fluorescence spectrum 63. NAD(P)H fluorescence images (2000 ms) for untreated HeLa cells as well treated cells after 15 min, 30 min, 60 min, and 180 min of treatment were captured. Images were processed for brightness and contrast (Fiji, www.fiji.sc). Standard workflow for image‐wide cell segmentation and calculation of mean intensity from objects was followed using CellProfiler (www.cellprofiler.org) to count number of cells and quantify autofluorescence intensities.

Larvae at 3 dpf were settled on a glass-bottom dish, and imaging was performed at 28 °C. Time-lapse fluorescence images in the head mesenchyme were acquired with a laser-scanning confocal microscope (LSM710; Zeiss) with a Plan-Apochromat 20×/0.8 M27 objective. Neutrophil motility at the CHT was imaged using a Zeiss EC Plan-NEOFLUAR 10×/0.3 objective. For neutrophil nucleus and cytosol reporter line imaging, an LD C-Apochromat 40×/1.1 W Korr M27 objective was used. The green and red channels were acquired sequentially with 0.1 ∼ 0.3% power of the 488-nm laser and 0.5 ∼ 2% power of the 561-nm laser, respectively, with a 200-μm pinhole at a speed of 1.27 μs per pixel and averaged (line 2). The fluorescent stacks were flattened using the maximum intensity projection and overlaid with a single slice of the bright-field image. Neutrophil chemotaxis upon LTB4 treatment was captured with a Zeiss ZV16 dissection microscope at a magnification of 2× every 15 s for 30 min.

Tissue sections (4 μm) were cut from paraffin-blocked duodena and mounted on glass slides, dewaxed with xylene, and then rehydrated with a series of alcohols. For antigen retrieval, sections were incubated in 10 mmol/L citrate buffer pH 6.0 containing 0.05% Tween-20, heated to boiling, and maintained at 95°C for 20 minutes. Sections were then incubated in 50 mmol/L glycine/NH₄Cl for 15 minutes at room temperature to prevent endogenous fluorescence, blocked for 1 hour at room temperature with 10% fluorescence dilution buffer (1 mmol/L CaCl₂, 1 mmol/L MgCl₂, 5% goat serum, 5% fetal bovine serum, 2% bovine serum albumin, PBS), and incubated with FPN1 rabbit polyclonal primary antibody EB9 (1/500 dilution) overnight at 4°C. Subsequent to washing with PBS, the sections were incubated with secondary antibody (Molecular Probes goat anti-rabbit Alexa Fluor A594; Life Technologies, Carlsbad, CA; cat #A-11012, 1/500 dilution) for 30 minutes, washed with PBS, and incubated with DAPI counterstain for 5 minutes at room temperature. After further PBS washes, the sections were mounted in Prolong Gold antifade mountant (Life Technologies). Sections were viewed with a Zeiss LSM780 with AxioObserver confocal microscope with a Plan-Apochromat ×20/0.8 M27 objective, and images were acquired and analyzed using the Zeiss Zen software.