Tcs spe confocal microscope

Fungal tissue was cleared using the MiPACT procedure as described previously with minor modification^{30 (link)}. Briefly, samples were grown for 48 h, and whole co-colonies cut from their growth medium, removing as much agar as possible. Samples were incubated at 4 °C overnight in 3% (v/v) paraformaldehyde. Samples were cleared for 2 weeks in a spinning 8% (w/v) SDS solution at 37 °C. Samples were washed in PBS, treated with 1 mg ml⁻¹ lysozyme for 30 minutes at 30 °C before being hybridized with HCR 2.0 eubacterial probes and incubated overnight at 46 °C while gently shaking. Samples were washed and the amplification step was performed using hairpins tagged with an AlexaFluor 647 fluorophore for visualization^{62 (link)}. Samples were stained with 1 μg ml⁻¹ DAPI, and microscopy was performed on an inverted confocal Leica model TCS SPE confocal microscope with a 10x objective for the colony center and edge, and a Nikon Ti2 Eclipse widefield microscope with a 4x objective for images of the colony ridge. Contrast of the HCR generated images were normalized to the brightest signal in like samples (i.e. colony edge vs edge, center vs center). Contrast was adjusted independently for each DAPI stained images for clarity of fungal morphology present near the bacteria. Images were analyzed in using Fiji^{63 (link)}.