Bx51 fluorescence microscopy system

At euthanization, kidneys were collected, cut sagittally into halves, and fixed in 10% buffered formalin solution (Sigma, St. Louis, MO, USA) for 48 h. Following fixation, kidney halves were washed in 100% ethanol and embedded in paraffin. Kidney halves were cut into 5–7 μm sections, which were deparaffinized, rehydrated, and permeabilized with 0.1% Triton solution (BioRad, Hercules, CA, USA). Tissue was blocked with 10% AquaBlock solution (EastCoastBio, North Berwick, ME, USA) before being immunolabeled with the lymphatic endothelial cell markers LYVE-1 or podoplanin (goat polyclonal; R&D Systems, Minneapolis, MN, USA) by overnight incubation at 4 °C. Alexafluor 488 or 594 (Life Technologies, Carlsbad, CA, USA) secondary antibodies were used to visualize lymphatic vessels. Samples were incubated with appropriately conjugated secondary antibodies for an hour at room temperature. Negative controls were incubated with only a secondary antibody. All labeled slides were mounted with ProLong Gold anti-fade reagent containing DAPI (Invitrogen, Carlsbad, CA, USA). An Olympus BX51 fluorescence microscopy system with Olympus Q5 camera was used for imaging. Images were captured at 40× magnification using Olympus CellSens imaging software (Olympus, Shinjuku, Tokyo, Japan). The same methods were utilized to image lymphatics in the heart, liver, lung, and skin (10× or 20× magnification).

To localize GFP-fused proteins using microscopy, strains were inoculated into 1 × VMM with 2% glucose and grown for 16 h at 45 °C. Microscopic observations were performed using an Olympus BX51 fluorescence microscopy system and images were processed using ImageJ software.

To localize GFP fusion proteins by microscopy, Cas9-gfp-positive transformants were inoculated in liquid MM containing 2% sucrose as the carbon source and grown for 24 h at 45 °C. Before imaging, hyphae were harvested and incubated with 1 mg mL⁻¹ 4′, 6-diamidino-2-phenylindole for 15 min. The microscopic observation was performed using an Olympus BX51 fluorescence microscopy system, with the ImageJ software used for image processing.

To localize GFP fusion proteins via microscopy, the M. thermophila strains in liquid MM medium were grown for 16 h at 45°C. The hyphae were harvested, washed several times with Vogel’s salts medium, transferred to inducing medium containing 2% (wt/vol) Avicel or glucose, and then grown for another 24 h. Before imaging, the hyphae were incubated with 1 mg mL⁻¹ DAPI for 15 min. The hyphae were examined using the Olympus BX51 fluorescence microscopy system and images were processed using ImageJ software.