Fluoview 1000 spectral confocal

Live imaging of the vasculature of Botryllus colonies labeled with BSA-Alexa 594 (A13101; Thermo Fisher Scientific, Waltham, MA) was carried out using a motorized fluorescence stereomicroscope MZ16FA (Leica, Germany) as previously described (Braden et al., 2014 (link)). Fixed tissue for in situ hybridization was imaged on an Olympus FluoView 1000 Spectral Confocal (Tokyo, Japan). Lipophilic staining of the vasculature labeled with CellMask Green (A37608; Thermo Fisher Scientific). Time-lapse movies (n = 5) taken as z-stacks were acquired with a Zeiss LSM 880 with Airyscan capabilities.

Human fibronectin at 150 μg mL^–1 in DPBS was added to glass confocal dishes and incubated for 2 h at 37 °C to improve its cell adhesion properties. Wells were washed 3 times with DPBS. Macrophages with particles were plated in the glass confocal dishes at 62 500 cells per cm² and incubated for 8 h in DMEMpy. Cells were incubated with l-NAME at 75 μg mL^–1 in DMEMpy for 1 h to inhibit biological NO production from nitric oxide synthases. Wells were aspirated and 9.3 μM 4-amino-5-methylamino-2′,7′-difluoroflurescein (DAF-FM-2DA), a dye that detects intracellular NO release, with l-NAME in DMEMpy was added to the cells and incubated for 1 h. Wells were washed five times with phenol red free, FBS free DMEMpy with l-NAME to remove uninternalized DAF-FM. Wells were exposed to a 794 nm laser for 90 s at 13.1 W cm^–2. NucBlue® was added to the wells to label cell nuclei. Wells were imaged 20 min later. Images were taken with an Olympus Fluoview 1000 Spectral Confocal. Care was taken to image cells as quickly as possible to prevent the release of extra NO. Samples were compared to macrophages exposed to the laser without particles.

S. cerevisiae CKY263 cultures were propagated overnight in 5 ml of synthetic complete media (SD-SCAA) lacking uracil, and supplemented with 2% galactose to induce expression of the GFP-tagged protein constructs. ∼10⁶ cells were pelleted and the mitochondria stained with Mito-ID Red detection reagent diluted 1:2500 (Mito-ID Red detection kit - Cat# ENZ-51007-500, Enzo Life Sciences, Ann Arbor, MI) according to manufacturer instructions before being affixed to polylysine-coated slides for visualization (Mazia et al., 1975 (link)). Mitochondria visualization and protein localization were determined by confocal microscopy with an Olympus Fluoview 1000 Spectral Confocal with a 600x objective (Olympus America, Center Valley, PA). Protein expression was detected via green fluorescence (excitation=488 nm, emission=510 nm) while mitochondria were stained to fluoresce red (excitation=559 nm, emission=598 nm). All samples were visualized at similar gain, PMT voltage, and magnification to directly compare fluorescence levels.