Rhodamine isothiocyanate

Adult CBA/BL6 mice and 5-day-old rat pups were euthanized by CO₂ inhalation, decapitated, and mouse cochleas and rat brains were removed and fixed by immersion in 4% paraformaldehyde/1% glutaraldehyde for 24 h. Paraformaldehyde-fixed Casper mutant zebrafish (Danio rerio) 6-8 weeks old were shipped to the University of Minnesota for TSLIM imaging. All specimens (except rat brains) were decalcified in a 10% solution of disodium ethylene diaminetetraacetic acid for 3 d and bleached in a 5% solution of H₂O₂ for 24 h. Mouse cochleas were separated from surrounding tissues and rat brains were hemisected and cut into thirds. The anterior 5 mm of the zebrafish head containing the brain and inner ears were used for imaging. All tissues were dehydrated in ascending concentrations of ethanol, immersed in hexane, and then cleared to transparency using Spalteholz fluid (9 ) which consists of 5:3 methyl salicylate:benzyl benzoate. Rat brains were cleared in 2:1 benzyl benzoate:benzyl alcohol (8 ) as this solution appeared to clear brain tissue better than Spalteholz fluid. The refractive index of the cleared specimens and the fluid-filled specimen chamber was ~1.56. Tissue fluorescence, which is necessary for TSLIM imaging, was induced either by chemical fixation autofluorescence (paraformaldehyde/glutaraldehyde), or by immersion in Rhodamine B isothiocyanate (1 mg/200 mL in Spalteholz for 24 h). A 590-nm bandpass filter was used to block scattered laser light from entering the CCD camera which was used to capture images.
Design and specifications of the TSLIM device are shown in computer-aided design (CAD) diagrams (Figure 1, Supplementary Figure 2). TSLIM consists of five primary components: two thin-sheet illuminators with aberration corrected objectives, a specimen chamber, a microscope with digital camera, motorized micropositioners and rotating stage, and control and imaging software. A complete list of materials used for the TSLIM device can be found in Supplementary Table 1 and a parts diagram is outlined in Supplementary Figure 2. Assembly and alignment procedures are also available in the Supplementary Materials. TSLIM contains two opposing laser illuminators that are mounted on a horizontal optical bench rail, which project their light sheets into the specimen chamber. Each illuminator consists of a 15 mW, green (λ = 532 nm) frequency-doubled Nd:YAG laser, a 10× or 20× Galilean beam expander, a cylindrical lens, and a 5× microscope objective. A 532-nm solid-state laser was selected because it excites and causes emission of a wide variety of fluorescent markers that are used for biological research. The laser beam is expanded and collimated using a Galilean beam expander, and then travels through a cylindrical lens, which focuses the beam in the y direction. The cylindrical lens and microscope objective assemble a Keplerian beam expander, which means that the beam leaving the microscope objective is collimated in the y direction. As the cylindrical lens does not affect the z component of the beam, the microscope objective has a focusing effect on the beam that results in a diffraction-limited light-sheet thickness in the z direction. The improvement in image quality by the addition of an objective lens is shown in Supplementary Figure 3; this was first used by Greger et al. (10 ). The light sheet then passes through the specimen chamber, which is positioned orthogonal to the optical axis of a horizontally mounted, Olympus MVX10 microscope (Olympus America, Inc., Center Valley, PA, USA). A glass cuvette or a custom-designed specimen chamber with an open top is filled with clearing fluid and the specimen is attached to a black Delrin rod (Small Parts Inc., Miramar, FL, USA) that extends into the middle of the chamber. The specimen attaching rod is connected to an optional, motorized rotating stage for convenient rotation/orientation of the specimen. The light sheet enters and leaves the chamber through the side windows and the fluorescent image plane in the tissue is viewed through the back window of the chamber nearest the MVX10 objective. Micropositioners (Newport Corp., Irvine, CA, USA) move the specimen (not the chamber) in the x,y,z directions (QImaging, Surrey, BC, Canada) through the illumination plane and at the focal point of the microscope objective. A custom LabVIEW program (version 8.6; National Instruments, Austin, TX, USA) was used to control the micropositioners and collect images using a Retiga 2000 (1600 × 1200 px) digital camera attached to the MVX10 microscope. Micropositioner control, image stitching and stack collection were automated and run on a Windows XP-based PC (See program flowchart in Supplementary Figure 4). The program controlled x-axis micropositioner movement while building a composite image from columns collected at each x-axis step. Column width was chosen to coincide with the confocal parameter of the light sheet and supplied to the CCD camera as a region of interest (ROI). After saving each composite image, the z axis was incremented and the next optical section was generated. Images were processed in Adobe Photoshop (Adobe CS3; Adobe Systems Incorporated, San Jose, CA, USA) and ImageJ (version 1.41; National Institutes of Health, Bethesda, MD, USA). After processing, stacks were loaded into Amira software (Visage Imaging Inc., Carlsbad, CA, USA) for reconstruction of individual tissue structures. See Supplementary Materials for information regarding obtaining a copy of our custom LabVIEW program or TSLIM community resources.

These assays were done essentially as described7 (link),8 (link),21 (link),46 (link)-48 (link), with some modifications. For in vitro recognition of zymosan and live fungal particles by macrophages, peritoneal exudate cells were isolated by lavage with ice-cold 5 mM EDTA in PBS from mice that had been treated intraperitoneally 4 d before with thioglycollate broth (Difco). The thioglycollate broth used does not contain yeast extract. Macrophages were plated in 24-well plates at a density of 2.5 × 10⁵ cells per well (for zymosan-recognition assays) or 1 × 10⁶ cells per well (for live C. albicans–recognition assays) in RPMI medium with 10% (volume/volume) heat-inactivated FCS. Cells were washed three times with medium before the addition of yeast particles. Fluorescein isothiocyanate–labeled zymosan (Invitrogen) or Rhodamine Green-X–labeled live C. albicans SC5314 were used in recognition assays at macrophage/particle ratios of 25:1 for zymosan and 10:1 to 0.04:1 for live C. albicans. After incubation for 30 min at 37 °C to allow efficient recognition of the fungal particles by both isoforms of dectin-1 (ref. 47 (link)), unbound yeast cells were washed away by four washes with RPMI medium plus 10% (volume/volume) heat-inactivated FCS. The medium was replaced and cells were cultured for 3 h at 37 °C for analysis of production of proinflammatory cytokines or for 20 h at 37 °C for measurement of IL-12p40 and IL-10. After incubation, supernatants were stored at −80 °C until use, cells were lysed in 3% (volume/volume) Triton X-100, pH 7.5, and fluorescence was measured with a Titer-Tek Fluoroskan II (Labsystems). Inflammatory responses to A. fumigatus (multiplicity of infection, 25:1) were measured after 20 h of fungus–macrophage coculture, as described11 (link).
For in vitro recognition of zymosan by neutrophils, peritoneal inflammatory cells were collected with 5 mM EDTA in PBS at 16–18 h after thioglycollate administration. Cells were pretreated for 30 min on ice with 100 μg/ml of β-glucan. Inflammatory cells (5 × 10⁵) were mixed with fluorescein isothiocyanate–labeled zymosan particles (1 × 10⁶ particles; 2:1 zymosan/inflammatory cell ratio) in 100 μl of RPMI medium plus 10% (volume/volume) heat-inactivated FCS in 5-ml polystyrene tubes and were centrifuged at 350g for 5 min at 4 °C. Inflammatory cells and zymosan were then incubated for 1 h at 37 °C. Cellular recognition of zymosan was determined by flow cytometry. Where required, zymosan was opsonized with complement as described above.
For in vitro recognition of zymosan by bone marrow–derived dendritic cells, the cells were incubated with various concentrations of unlabeled zymosan (Sigma) for 20 h at 37 °C, then supernatants were collected and were stored at −80 °C until use for cytokine analysis.