From e843eb50c9d803f1bfc8f0806551d9b662825e09 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Mon, 8 Jan 2024 19:05:32 +0100
Subject: [PATCH] add assay StainingForConfocalMicroscopy

---
 .../isa.assay.xlsx                            | Bin 6914 -> 6914 bytes
 .../isa.assay.xlsx                            | Bin 6915 -> 6915 bytes
 assays/MALDI-TOFAnalysis/isa.assay.xlsx       | Bin 6900 -> 6900 bytes
 .../isa.assay.xlsx                            | Bin 7020 -> 7020 bytes
 assays/OxidativeBurstAssay/isa.assay.xlsx     | Bin 6899 -> 6899 bytes
 assays/ProteinPull-down/isa.assay.xlsx        | Bin 6898 -> 6898 bytes
 .../isa.assay.xlsx                            | Bin 6910 -> 6910 bytes
 .../StainingForConfocalMicroscopy/README.md   |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6908 bytes
 .../protocols/.gitkeep                        |   0
 .../protocols/HordeumVulgare.md               |   4 ++++
 .../protocols/RootStainingOfRIrregularis.md   |   2 ++
 assays/qRT-PCR/isa.assay.xlsx                 | Bin 6893 -> 6893 bytes
 isa.investigation.xlsx                        | Bin 10519 -> 11000 bytes
 .../isa.study.xlsx                            | Bin 7536 -> 7571 bytes
 .../protocols/RootStainingOfRIrregularis.md   |   2 --
 studies/HordeumVulgare/isa.study.xlsx         | Bin 7808 -> 7539 bytes
 .../StainingForConfocalMicroscopy.md          |   4 ----
 studies/NicotianaBenthamiana/isa.study.xlsx   | Bin 7582 -> 7581 bytes
 20 files changed, 6 insertions(+), 6 deletions(-)
 create mode 100644 assays/StainingForConfocalMicroscopy/README.md
 create mode 100644 assays/StainingForConfocalMicroscopy/dataset/.gitkeep
 create mode 100644 assays/StainingForConfocalMicroscopy/isa.assay.xlsx
 create mode 100644 assays/StainingForConfocalMicroscopy/protocols/.gitkeep
 create mode 100644 assays/StainingForConfocalMicroscopy/protocols/HordeumVulgare.md
 create mode 100644 assays/StainingForConfocalMicroscopy/protocols/RootStainingOfRIrregularis.md
 delete mode 100644 studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md
 delete mode 100644 studies/HordeumVulgare/protocols/StainingForConfocalMicroscopy.md

diff --git a/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx b/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx
index 2f818eb899f4047896ead63b154b2cc4f9ecbf52..228d5fa5caeeb72700819e39d89d9673086696a1 100644
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diff --git a/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx b/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx
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diff --git a/assays/MALDI-TOFAnalysis/isa.assay.xlsx b/assays/MALDI-TOFAnalysis/isa.assay.xlsx
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diff --git a/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx b/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx
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diff --git a/assays/OxidativeBurstAssay/isa.assay.xlsx b/assays/OxidativeBurstAssay/isa.assay.xlsx
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diff --git a/assays/ProteinPull-down/isa.assay.xlsx b/assays/ProteinPull-down/isa.assay.xlsx
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diff --git a/assays/StainingForConfocalMicroscopy/protocols/.gitkeep b/assays/StainingForConfocalMicroscopy/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/StainingForConfocalMicroscopy/protocols/HordeumVulgare.md b/assays/StainingForConfocalMicroscopy/protocols/HordeumVulgare.md
new file mode 100644
index 0000000..2d98a76
--- /dev/null
+++ b/assays/StainingForConfocalMicroscopy/protocols/HordeumVulgare.md
@@ -0,0 +1,4 @@
+## 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.
\ No newline at end of file
diff --git a/assays/StainingForConfocalMicroscopy/protocols/RootStainingOfRIrregularis.md b/assays/StainingForConfocalMicroscopy/protocols/RootStainingOfRIrregularis.md
new file mode 100644
index 0000000..f0a6433
--- /dev/null
+++ b/assays/StainingForConfocalMicroscopy/protocols/RootStainingOfRIrregularis.md
@@ -0,0 +1,2 @@
+## 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.
\ No newline at end of file
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diff --git a/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.study.xlsx b/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.study.xlsx
index d252a07f4395555e72e5409b4037de3e85aadb62..80acb6d1dc4685e2c6559ed5cfd159b0f91478f9 100644
GIT binary patch
delta 2254
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diff --git a/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md b/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md
deleted file mode 100644
index 7b96001..0000000
--- a/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md
+++ /dev/null
@@ -1,2 +0,0 @@
-## 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.
\ No newline at end of file
diff --git a/studies/HordeumVulgare/isa.study.xlsx b/studies/HordeumVulgare/isa.study.xlsx
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diff --git a/studies/HordeumVulgare/protocols/StainingForConfocalMicroscopy.md b/studies/HordeumVulgare/protocols/StainingForConfocalMicroscopy.md
deleted file mode 100644
index bd9785d..0000000
--- a/studies/HordeumVulgare/protocols/StainingForConfocalMicroscopy.md
+++ /dev/null
@@ -1,4 +0,0 @@
-## 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.
\ No newline at end of file
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