Root fragments and nodules were fixed as mentioned above. After that, they were washed with 0.1 M phosphate buffer three times for 15 min each, once with water for 15 min, and dehydrated for 10 min in 10%, 30%, 50%, 70%, 90%, and 100% (v/v) ethanol, and sequentially embedded in plastic Technovit 7100 (Heraeus Kulzer). Sections of 5 μm were made using a microtome (RJ2035, Leica), stained with 0.05% Toluidine Blue (Sigma), mounted in Euparal (Carl Roth), and analyzed with a Leica AU5500B microscope equipped with a DFC425c camera (Leica). Transgenic pMtHDT::GFP::MtHDT nodules and root segments were sectioned into 60-µm slices by vibratome (VT1000, Leica) and stained with propidium iodide for 2 min. Sections were then washed three times in phosphate buffer containing triton x-100 and mounted on slides with MQ water. All confocal images were acquired using Leica SP8 confocal laser scanning microscope (Leica, Germany). GFP and EdU signals were detected with excitation wavelength and detection windows of 488 and 500–530 nm, propidium iodide was detected with excitation wavelength and detection windows of 543 and 580–640 nm.
Au5500b microscope
Manufactured by Leica
The AU5500B is a high-performance microscope designed for laboratory applications. It features a range of advanced optical and mechanical components to provide clear, detailed images. The microscope's core function is to enable precise observation and analysis of samples at various magnification levels.
Automatically generated - may contain errors
Lab products found in correlation
Dfc425c camera, by Leica (5 mentions)
Invitrogen viewrna ish tissue 1 plex assay kit, by Thermo Fisher Scientific (3 mentions)
Toluidine blue, by Merck Group (2 mentions)
Rj2035, by Leica (2 mentions)
Technovit 7100, by Kulzer (2 mentions)
Sp8 laser scanning confocal microscope, by Leica (2 mentions)
Euparal, by Carl Roth (2 mentions)
Rj2035 microtome, by Leica (2 mentions)
Vt1000, by Leica (1 mentions)
6 protocols using au5500b microscope
1
Comprehensive Nodule Microscopy Analysis
2
Nodule and Root Histological Analysis
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Root fragments and nodules were fixed as mentioned above. After that they were washed with 0.1 M phosphate buffer 3 times for 15 min each, once with water for 15 min, and dehydrated for 10 min in 10%, 30%, 50%, 70%, 90% and 100% ethanol, and sequentially embedded in plastic Technovit 7100 (Heraeus Kulzer). Sections were made of 5μm using a microtome (RJ2035, Leica), stained with 0.05% Toluidine Blue (Sigma), mounted in Euparal (Carl Roth), and analysed with a Leica AU5500B microscope equipped with a DFC425c camera (Leica). Transgenic pMtHDT::GFP::MtHDT nodules and root segments were sectioned into 60µm slices by vibratome (VT1000, Leica) and mounted on slides with MQ water. All confocal images were acquired using Leica SP8 confocal laser scanning microscope (Leica, Germany). GFP and EdU signal were detected with an excitation wavelength of 488 nm and DsRed was detected with an excitation wavelength of 543 nm.
3
RNA In Situ Hybridization of Plant Nodules
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The nodules and nodule primordia were fixed with 4% (w/v) paraformaldehyde mixed with 5% (v/v) glutaraldehyde in 50-mM phosphate buffer (pH 7.4) and embedded in paraffin (Paraplast X-tra, McCormick Scientific). Sections of 7 μm were cut by RJ2035 microtome (Leica). RNA in situ hybridization was performed using Invitrogen ViewRNA ISH Tissue 1-Plex Assay kit (Thermo Fisher Scientific) according to the manual protocol (https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0018633_viewRNA_ISH_UG.pdf&title=VXNlciBHdWlkZTogVmlld1JOQSBJU0ggVGlzc3VlIEFzc2F5 ). RNA ISH probe sets were designed and produced by Thermo Fisher Scientific. Catalogue numbers of probe sets are the following: MtHDT1 is VF1-14234, MtHDT2 is VF1-18132, MtHDT3 is VF1-6000218, and MtHMGR1 is VF1-20373. Any probe set was omitted for a negative control. Slides were analyzed with an AU5500B microscope equipped with a DFC425c camera (Leica).
4
Visualizing Mycorrhizal Root Colonization
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Mycorrhized roots were first cleared in 10% potassium hydroxide at 90°C for 20 min. Roots were then washed twice with phosphate‐buffered saline (150 mM NaCl, 10 mM sodium hydrogen phosphate, and 1.8 mM KH2PO4, pH 7.4) followed by incubation in 0.2 µg ml−1 wheat germ agglutinin (WGA)‐Alexa Fluor® 488 in PBS at 4°C overnight. Microscopy was done using a Leica AU5500B microscope. Mycorrhization was quantified as previously described (Trouvelot et al., 1986 ).
5
RNA in situ Hybridization in Medicago
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RNA in situ hybridisation was conducted using Invitrogen ViewRNA ISH Tissue 1‐Plex Assay kits (Thermo Fisher Scientific) according to manufacturer's user guide and optimised for Medicago root and nodules sections (Kulikova et al., 2018 ). RNA ISH probe sets were designed and synthesised at Thermo Fisher Scientific. Catalogue numbers are VF1‐20312 for NIN (Medtr5g099060), VP2W7MP for CP2 (Medtr5g022560), VP7DPDU for SymCRK (Medtr3g079850), VPRWEMC for NAD1 (Medtr7g022640) and VF‐20311 for NF‐YA1 (Medtr1g056530). Each probe set was tested on tissue where it should not be present, in this case on noninoculated roots. As a negative control for each hybridisation procedure any probe set was omitted. In both cases no hybridisation signals were detected. The in situ images were taken with an AU5500B microscope equipped with a DFC425c camera (Leica).
6
RNA In Situ Hybridization of Nodule Transcripts
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The nodules and nodule primordia were fixed with 4% paraformaldehyde mixed with 5% glutaraldehyde in 50 mM phosphate buffer (pH 7.4) and embedded in paraffin (Paraplast X-tra, McCormick Scientific). Sections of 7 μm were cut by RJ2035 microtome (Leica). RNA in situ hybridisation was performed using Invitrogen ViewRNA ISH Tissue 1-Plex Assay kit (Thermo Fisher Scientific) according to the manual protocol (https://www.thermofisher.com/documentconnect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0018633_viewRNA_ISH_UG.pdf&title=VXNlciBHdWlkZTogVml ld1JOQSBJU0ggVGlzc3VlIEFzc2F5). RNA ISH probe sets were designed and produced by Thermo Fisher Scientific. Catalogue numbers of probe sets are the following: for MtHDT1 is VF1-14234, for MtHDT2 is VF1-18132, for MtHDT3 is VF1-6000218 and for MtHMGR1 is VF1-20373. Any probe set was omitted for a negative control. Slides were analysed with an AU5500B microscope equipped with a DFC425c camera (Leica).
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