From cbb1f646641d6ca31db31e2c4b17f4fb591ad324 Mon Sep 17 00:00:00 2001 From: Viktoria Petrova <vipet103@hhu.de> Date: Wed, 19 Jun 2024 21:43:40 +0200 Subject: [PATCH] redo staining isa assay --- .../isa.assay.xlsx | Bin 13305 -> 13305 bytes .../isa.assay.xlsx | Bin 17626 -> 17626 bytes .../isa.assay.xlsx | Bin 14945 -> 14945 bytes .../protocols/RhizophagusIrregularis.md | 6 +++++- .../isa.assay.xlsx | Bin 19957 -> 19958 bytes assays/MALDI-TOFAnalysis/isa.assay.xlsx | Bin 13056 -> 13057 bytes .../isa.assay.xlsx | Bin 10823 -> 10824 bytes .../MultipleSequenceAlignment/isa.assay.xlsx | Bin 6903 -> 6904 bytes assays/OxidativeBurstAssay/isa.assay.xlsx | Bin 14412 -> 14413 bytes .../isa.assay.xlsx | Bin 17443 -> 17444 bytes assays/ProteinPull-down/isa.assay.xlsx | Bin 20810 -> 20811 bytes .../isa.assay.xlsx | Bin 6910 -> 6911 bytes .../isa.assay.xlsx | Bin 6908 -> 10128 bytes .../protocols/RootStainingOfRIrregularis.md | 3 --- ... 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Germinated seedlings were transferred to 9 × 9 cm pots containing autoclaved 1:1 silica sand:vermiculite mixture inoculated with 700 mg *R. irregularis* spore inoculum (10,000 spores·g-1 moist diatomaceous earth powder [50% water]) (Symplanta, Darmstadt, Germany). Approximately 500 mg of the inoculum was evenly mixed into the lower two-third substrate layer, further 200 mg were evenly sprinkled into a hole in the upper third of the substrate layer, into which the seedlings were transplanted. The seedlings were grown in the greenhouse and watered weekly with 30 mL of deionized water and tap water in a 1:1 ratio. Roots were harvested at 28 dpi and stored in 50% EtOH at 4 °C until staining. \ No newline at end of file +Barley control and mutant seeds were sterilized and germinated as described previously (Mahdi et al., 2022). Germinated seedlings were transferred to 9 × 9 cm pots containing autoclaved 1:1 silica sand:vermiculite mixture inoculated with 700 mg *R. irregularis* spore inoculum (10,000 spores·g-1 moist diatomaceous earth powder [50% water]) (Symplanta, Darmstadt, Germany). Approximately 500 mg of the inoculum was evenly mixed into the lower two-third substrate layer, further 200 mg were evenly sprinkled into a hole in the upper third of the substrate layer, into which the seedlings were transplanted. The seedlings were grown in the greenhouse and watered weekly with 30 mL of deionized water and tap water in a 1:1 ratio. Roots were harvested at 28 dpi and stored in 50% EtOH at 4 °C until staining. + +## Staining + +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. 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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. 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