add assay StainingForConfocalMicroscopy

assays/StainingForConfocalMicroscopy/protocols/HordeumVulgare.md

+## Hordeum vulgare
+Root tissue of barley control and mutant plants colonized by *S. indica* was harvested at 6 dpi and then stained as previously described (Hilbert et al., 2019). Briefly, roots were incubated at 95 °C for 2 min in 10% KOH, washed 3 times for 30 min in deionized water and 3 times for 30 min in PBS (pH 7.4). The roots were stained for 5 min under vacuum and then washed three times with deionized water. Fungal structures were visualized using 10 μg·mL-1 fluorescently labeled wheat germ agglutinin (WGA-AF488, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) in PBS (pH 7.4) and imaging was conducted with an excitation wavelength of 488 nm and emission detection between 500-540 nm. Papillae and root cell wall appositions were stained with 10 μg·mL-1 fluorescently labeled Concanavalin A (ConA-AF633, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) in PBS (pH 7.4) and imaged by excitation at 633 nm and detection at 650-690 nm.
+Callose was stained by aniline blue according to a protocol adapted from Mason and coworkers (Mason et al., 2020). Roots were incubated at 95 °C for 2 min in 10% KOH, washed 3 times for 30 min in deionized water and 3 times for 30 min in PBS (pH 7.4). Roots were washed for 1 h at RT under continuous shaking in 67 mM K2HPO4 (pH 12). The samples were then incubated for 1 h at RT under continuous shaking in an aniline blue solution composed of 0.01% aniline blue (w/v) in 67 mM K2HPO4 (pH 12). Roots were washed again for 1 h at RT under continuous shaking in 67 mM K2HPO4 (pH 12). Imaging was conducted with an excitation wavelength of 405 nm and emission detection from 490-510 nm.
+Images were taken with a Leica TCS SP8 confocal microscope (Wetzlar, Germany). The percentage area of ConA staining was quantified using ImageJ81 in maximum intensity projections of 10-slice Z-stacks with an image depth of 10 μm. At least 24 different root regions of each genotype were analyzed.
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assays/StainingForConfocalMicroscopy/protocols/RootStainingOfRIrregularis.md

+## Root staining of *R. irregularis*
+Roots were stained according to a previously published protocol (Vierheilig et al., 1998). Briefly, roots were incubated for 15 min at 95 °C in 10% KOH, washed with 10% acetic acid and incubated for 5 min at 95 °C with a staining solution of 5% ink (Pelikan, Falkensee, Germany) in 5% acetic acid. After staining, the roots were carefully washed with tap water, then incubated in 5% acetic acid at 4 °C for at least 20 min. The ink-stained root tissue was cut into 1 cm segments with a scalpel and 30 segments of similar diameter were randomly selected from each genotype. Cross-section points were determined from 10 random cuts per root segment. Ink-stained *R. irregularis* structures such as intraradical hyphae (IRH), extraradical hyphae (ERH), arbuscules and vesicles were visualized with a light microscope (AxioStar, Carl Zeiss, Jena, Germany) at 10× magnification. Colonization with R. irregularis was scored as positive if IRH, arbuscules or vesicles were present. The roots of four biological replicates per genotype were examined.
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studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md

-## Root Staining
-Roots were stained according to a previously published protocol.83 Briefly, roots were incubated for 15 min at 95 °C in 10% KOH, washed with 10% acetic acid and incubated for 5 min at 95 °C with a staining solution of 5% ink (Pelikan, Falkensee, Germany) in 5% acetic acid. After staining, the roots were carefully washed with tap water, then incubated in 5% acetic acid at 4 °C for at least 20 min. The ink-stained root tissue was cut into 1 cm segments with a scalpel and 30 segments of similar diameter were randomly selected from each genotype. Cross-section points were determined from 10 random cuts per root segment. Ink-stained *R. irregularis* structures such as intraradical hyphae (IRH), extraradical hyphae (ERH), arbuscules and vesicles were visualized with a light microscope (AxioStar, Carl Zeiss, Jena, Germany) at 10× magnification. Colonization with R. irregularis was scored as positive if IRH, arbuscules or vesicles were present. The roots of four biological replicates per genotype were examined.
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studies/HordeumVulgare/protocols/StainingForConfocalMicroscopy.md

-## Staining for confocal microscopy
-Root tissue of barley control and mutant plants colonized by *S. indica* was harvested at 6 dpi and then stained as previously described.46 Briefly, roots were incubated at 95 °C for 2 min in 10% KOH, washed 3 times for 30 min in deionized water and 3 times for 30 min in PBS (pH 7.4). The roots were stained for 5 min under vacuum and then washed three times with deionized water. Fungal structures were visualized using 10 μg·mL-1 fluorescently labeled wheat germ agglutinin (WGA-AF488, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) in PBS (pH 7.4) and imaging was conducted with an excitation wavelength of 488 nm and emission detection between 500-540 nm. Papillae and root cell wall appositions were stained with 10 μg·mL-1 fluorescently labeled Concanavalin A (ConA-AF633, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) in PBS (pH 7.4) and imaged by excitation at 633 nm and detection at 650-690 nm.
-Callose was stained by aniline blue according to a protocol adapted from Mason and coworkers.89 Roots were incubated at 95 °C for 2 min in 10% KOH, washed 3 times for 30 min in deionized water and 3 times for 30 min in PBS (pH 7.4). Roots were washed for 1 h at RT under continuous shaking in 67 mM K2HPO4 (pH 12). The samples were then incubated for 1 h at RT under continuous shaking in an aniline blue solution composed of 0.01% aniline blue (w/v) in 67 mM K2HPO4 (pH 12). Roots were washed again for 1 h at RT under continuous shaking in 67 mM K2HPO4 (pH 12). Imaging was conducted with an excitation wavelength of 405 nm and emission detection from 490-510 nm.
-Images were taken with a Leica TCS SP8 confocal microscope (Wetzlar, Germany). The percentage area of ConA staining was quantified using ImageJ81 in maximum intensity projections of 10-slice Z-stacks with an image depth of 10 μm. At least 24 different root regions of each genotype were analyzed.
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