880 laser scanning confocal microscope

Cysts and transwells were fixed with 4% methanol-free formaldehyde for 60 min at room temperature. Cysts were washed and permeabilised by three 20 min washes in wash buffer (1.3 M NaCl, 132 mM Na₂HPO₄, 35 mM NaH₂PO₄, 75 mM NaN₃, 0.5% BSA, 2% Triton X-100, 0.4% Tween-20). Transwells were permeabilised in 0.3% Triton X-100 for 30 min and blocked in 5% BSA containing 15% FCS for 1 h. Next, cysts and transwells were incubated with primary antibodies diluted in 3% BSA in PBS overnight, followed by a 2 h secondary antibody incubation, and counterstained with the DNA dye Hoechst 33342 and Alexa Fluor 488/647 phalloidin. Confocal images were acquired on a Zeiss LSM 710, 780 or 880 confocal laser-scanning microscope. At least ten images were captured for each representative image depicted in the figures, and a minimum of three experiments was performed (n indicated in each figure).

Five-day-old pea aphids were fed for 24 h on artificial diets containing 8 µM of either PAF/GNA, PAF, GNA, or ovalbumin, or control (no protein) diet with PI counterstain (100 ng/µL) and subsequently chased with artificial diet and PI for 24 or 48 h. Aphid guts were dissected by submerging live adults in ice-cold PBS and fine tip forceps were used to gently separate the gut from the head and the body. Dissected guts were mounted directly onto glass slides in Vectashield H-100, overlaid with glass coverslips, and visualised using a Zeiss 880 confocal laser scanning microscope. Settings were as follows: Objective Plan-Apochormat 20x/0.8 M27; FITC excitation and image capture: 488 nm laser (0.5% power), wavelengths collected between 493 and 572 nm, gain 575; PI excitation and image capture: 543 nm laser (1.5% power), wavelengths collected between 584 and 735 nm, gain 800. To quantify the fluorescence in each treatment a minimum of 6 guts from each condition were imaged utilising the same laser and image settings. The outline of the PI counterstain was traced in FIJI, an ImageJ based analysis software and the intensity of the green channel measured using the pixel intensity measuring tool in the base software package [40 (link), 41 (link)]. The mean intensity of the traced area was then averaged, and the standard error calculated.

In vitro FRAP assays were performed with a Zeiss 880 Confocal Laser Scanning Microscope equipped with a 100×, 1.4 NA oil immersion objective. Samples were prepared as described above. All in vitro FRAP experiments used the 488 nm laser at 100% for bleaching conditions. For N-polyC at 150 mM NaCl, 5 bleaching iterations were used, and images were taken every 2 seconds for 232 seconds. For viral RNA at 150 mM NaCl, 100 bleaching iterations were used, and images were taken every 10 seconds for 8 minutes. For the experiments conducted at 50 mM NaCl, droplets containing polyC RNA or viral RNA were bleached with 20 iterations, and images were taken every 5 seconds for 5 minutes. For droplets containing N protein only, 20 bleaching iterations were used, and images were collected every 2 seconds for 6.5 minutes. All data were acquired using Zeiss Zen 2.3 SP1 FP3 (black) (64bit) Version 14.0.20.201. Data were background corrected, converted to normalized intensity, and fit to an exponential decay function in Origin.

In vivo imaging and FRAP measurements were performed with a Zeiss 880 Confocal Laser Scanning Microscope equipped with a 63×, 1.4 NA oil immersion objective. Samples were prepared as described above. All in vivo FRAP experiments used the 488 nm laser at 100% for bleaching conditions. Cells expressing N-GFP, ΔR2-GFP, or GFP only were bleached with 30 iterations, and images were collected continuously at 6.7 Hz for 30 seconds. All data were acquired using Zeiss Zen 2.3 SP1 FP3. Data were converted to the normalized intensity and fit to an exponential decay function in Origin.

Intestinal endothelial cells from mT/mG pups were purified as previously described^{50 (link)}. Myeloid cells were purified from either Cx3cr1-GFP or wild-type (WT) pup intestinal tissues. All cell isolations were performed by pooling 8–15 pup intestines in each group to yield enough cells for experiments. In all, 4 × 10⁴ endothelial cells and 2 × 10⁴ myeloid cells or macrophages were cultured on Matrigel separately or together, in endothelial culture media without or with IGF-1 (100 ng/ml) and/or PPP (500 nM). Cell sprouting images were taken by a Zeiss 880 Confocal Laser Scanning Microscope at 10x magnification. The length of the sprouts from each image was measured using photoshop. Alternatively, isolated macrophages and endothelial cells were cultured in vitro either separately or together at a 1:2 ratio in triplicates in 24-well plates. Cells were collected after 48 h of culture and stained with indicated markers or isotype control antibodies for flow cytometric analysis after gating on live singlet cells. IGF-1 was stained after cell fixation and permeabilization using eBioscience™ Intracellular Fixation & Permeabilization Buffer (88-8824, Thermo Fisher) post cell surface marker staining. Endothelial cells were identified as Tie-2⁺CX3CR1⁻ population since the CD31 marker could not be used, being no longer detectable after cells have been cultured in vitro and retrieved.