Texas red conjugated dextran

Paracellular barrier function of tracheal SJs was assayed as previously described by injecting a 10 kD Texas Red-conjugated dextran (Molecular Probes) into the body cavities of stage 16 embryos [75] (link). Injected embryos were imaged on a Leica TCS SP2 confocal microscope within 20 min of injection.

Cold anesthetized animals with fluorescent labeled ALs (partial Orco-Gal4/UAS-DsRed) were mounted with their ventral side pointing upside down with dental wax on a microscope slide. The pronotum and the head capsule were opened using a piece of a razor blade held by a blade breaker, with two parallel longitudinal cuts along the compound eyes. The cuticle, fat tissue, and tracheae were removed. Afterwards, the head capsule and pronotum were covered with ringer solution [197 (link)]. A tungsten needle was sharpened in 2 M KOH with 5–8 volts as described in [198 ], followed by coating with Texas Red conjugated dextran (3000 MW; Molecular Probes, Invitrogen) dissolved in NGS and air-dried. The injection of dye into the DsRed-labeled AL was performed manually under a fluorescence stereomicroscope (SteREO Lumar.V12, Carl Zeiss MicroImaging, Jena, Germany) by careful perforation. The treated animals were kept in a moist chamber for about 1 h at room temperature to let the dye diffuse. Afterwards, the brains were dissected, fixed, washed, and pre-incubated with NGS as described previously and afterwards incubated with Alexa Fluor 488-coupled phalloidin (1/200), DAPI (1/20,000), and 2 % NGS for 2 days at 4 °C. Subsequently, the brains were washed, dehydrated, cleared, and mounted in Permount as described above.

w^m4h; CyORoi/Sco 3h starved flies were allowed to lay eggs for 50 minutes on apple juice agar plates with yeast. Approximately 100–150 decorionated eggs were injected with 1–2 pl of an injection solution containing 5 mM NaCl, 5% Texas Red conjugated dextran (Molecular Probes), and 1 ng/μl of total RNA extracted and purified from testis of either Δw or w^seyDrosophila males.

GFP-expressing bacteria were added to P. tetraurelia at MOI = 20,000, which were then incubated at 25°C for 30 min to 48 h. P. tetraurelia was fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Images of fluorescence were obtained using a FluoView FV100 confocal laser scanning microscope (Olympus, Tokyo, Japan).
When LysoTracker (Life Technologies, Carlsbad, CA, United States) was used, P. tetraurelia was fixed 30 min after infection. After fixation, samples were washed twice with PBS and then incubated with LysoTracker (50 nM) for 30 min. LysoTracker-positive LCVs were counted using microscopy, and the data are shown as an average of three fields.
When a Texas Red-conjugated dextran (TRDx, Thermo Fisher Scientific, MA, United States) was used, samples were washed 1 h after infection to remove extracellular bacteria using a nylon mesh as described above. TRDx (50 μg/mL) was added to P. tetraurelia. At each sampling time, P. tetraurelia was fixed and washed twice with PBS. The number of TRDx-containing vacuoles in individual P. tetraurelia was counted and expressed as the average of 30 P. tetraurelia cells.

Female tumour-bearing mice between 12–14 weeks of age were injected with 25 μg kg⁻¹ Texas red-conjugated dextran (70,000 MW; Thermofisher Scientific, Waltham, MA) 30 min prior to euthanasia. Tissues were embedded in OCT (Electron Microscopy Sciences, Hatfield, PA), cut into 5 μm sections and imaged on an Olympus IX71 inverted multicolour fluorescent microscope.