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).
Rj2035 microtome
Manufactured by Leica
The RJ2035 is a microtome, a precision instrument used for cutting thin slices or sections of materials, such as biological samples, for microscopic examination. The core function of the RJ2035 microtome is to produce consistent, high-quality tissue sections with uniform thickness.
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)
Dm5500b microscope, by Leica (3 mentions)
Au5500b microscope, by Leica (2 mentions)
Technovit 7100, by Kulzer (2 mentions)
Vt1000s vibratome, by Leica (1 mentions)
Axio imager a1 microscope, by Zeiss (1 mentions)
Os140box, by Duchefa Biochemie (1 mentions)
Syto13, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica (1 mentions)
Toluidine blue o, by Merck Group (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Calcofluor white, by Leica (1 mentions)
Lr white, by Electron Microscopy Sciences (1 mentions)
7 protocols using rj2035 microtome
1
RNA In Situ Hybridization of Plant Nodules
2
In Situ Hybridization of Medicago Roots
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Medicago roots hosting the AM fungus were fixed with 4% paraformaldehyde mixed with 3% glutaraldehyde in 50 mM phosphate buffer (pH 7.4) overnight, dehydrated in serial dilution of ethanol and then embedded in paraffin (Paraplast X‐tra; McCormick Scientific, St Louis, MO, USA) as described (Kulikova et al., 2018 ). Next, 7‐μm‐thick root sections were cut using an RJ2035 microtome (Leica). RNA in situ hybridisation was conducted using Invitrogen ViewRNA ISH Tissue 1‐Plex Assay kits (Thermo Fisher Scientific, Waltham, MA, USA) according to the user manual available online (shorturl.at/fuzPT).
RNA ISH probe sets were designed and synthesised by request at Thermo Fisher Scientific. A typical probe set consisted of c. 20 synthetic adjacent oligonucleotide pairs. Each of these pairs was composed of a 20‐bp primary sequence designed to target specific regions across the target mRNA sequence and a secondary extended sequence serving as a template for the amplification and detection of hybridisation signals. A probe set for RiNLE1 (catalogue number VF1‐6001202) covered the mRNA sequence from 2 bp to 613 bp and a probe set for MtPT4 (VF1‐19337) covered the region from 390 bp to 1296 bp. A probe set for MtPT4 was used as a positive control for in situ hybridisation. As a negative control, a RiNLE1 sense probe set (VPEPR3M) was used and probe sets were omitted for hybridisation.
RNA ISH probe sets were designed and synthesised by request at Thermo Fisher Scientific. A typical probe set consisted of c. 20 synthetic adjacent oligonucleotide pairs. Each of these pairs was composed of a 20‐bp primary sequence designed to target specific regions across the target mRNA sequence and a secondary extended sequence serving as a template for the amplification and detection of hybridisation signals. A probe set for RiNLE1 (catalogue number VF1‐6001202) covered the mRNA sequence from 2 bp to 613 bp and a probe set for MtPT4 (VF1‐19337) covered the region from 390 bp to 1296 bp. A probe set for MtPT4 was used as a positive control for in situ hybridisation. As a negative control, a RiNLE1 sense probe set (VPEPR3M) was used and probe sets were omitted for hybridisation.
3
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).
4
Root Tissue Fixation, Sectioning, and Imaging
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Samples were fixed under vacuum for 48 h with 4% paraformaldehyde (w/v) and 5% glutaraldehyde (v/v) in 50 mM phosphate buffer, pH 7.2. Tissue processing and embedding for plastic and paraffin sectioning were performed as described by Long et al. (2015) (link). Root sections were made using an RJ2035 microtome (Leica Microsystems), stained in ruthenium red for 10 min, and then mounted in Depex. Images were captured with a Normaski microscope (Axio Imager; DM5500B microscope, Carl Zeiss).
5
Microscopic Analysis of Root Anatomy
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For microscopy studies, root segments were all collected at 6 DAG. For measuring the length of the RAM, ~ 5 mm root tips were cut and immersed in chloral hydrate solution at 4 °C overnight, analyzed under Axio Imager A1 microscope (Zeiss) with Nomarski optics. For measuring cross-sectional areas, root segments were fixed with 4% paraformaldehyde (w/v), 5% glutaraldehyde (v/v) in 0.05 M sodium phosphate buffer (pH 7.2) at 4 °C overnight. The fixed material was dehydrated in an ethanol series and subsequently embedded in Technovit 7100 (Heraeus Kulzer) according to the manufacturer’s protocol. Sections (5 μm) were made with a RJ2035 microtome (Leica Microsystems) stained 1.5 min in 0.05% toluidine blue O. For phloroglucinol-HCl staining, root segments were fixed as abovementioned. The fixed material was washed with 1 x PBS (sodium phosphate buffer), and directly embedded in 6% low melting agarose dissolved in 1 x PBS. Sections (50 μm) were made with a VT1000 S vibratome (Leica Microsystems), stained with 2% phloroglucinol (in 95% ethanol) for 2 min and applied with a few drops of 37% HCl. Sections were all analysed by using a DM5500B microscope equipped with a DFC425C camera (Leica Microsystems).
6
Tissue Culture and Nodulation Assay
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Rooted tissue culture plantlets for phenotyping assays were grown in crystal-clear polypropylene containers (1 L) with a gas-exchange filter (OS140BOX; Duchefa Biochemie). Pots were half-filled with Agroperlite (Maasmond-Westland) and watered with modified EKM medium (3 mm MES [C6H13NO4], pH 6.6, 2.08 mm MgSO4, 0.88 mm KH2PO4, 2.07 mm K2HPO4, 1.45 mm CaCl2, 0.7 mm Na2SO4, 0.375 mm NH4NO3, 15 μm iron citrate, 6.6 μm MnSO4, 1.5 μm ZnSO4, 1.6 μm CuSO4, 4 μm H3BO3, and 4.1 μm Na2MoO4; Becking, 1983 ). For nodulation assays, modified EKM medium (Becking, 1983 ) was inoculated with rhizobia (OD600 = 0.025) prior to planting the shoots. For inoculation with strain S. fredii NGR234.pHC60, containers were half-filled with sterilized river sand and watered with modified EKM medium containing the bacteria at OD600 = 0.05.
All nodules were fixed in buffer containing 4% (w/v) paraformaldehyde mixed with 3% (v/v) glutaraldehyde in 50 mm phosphate (pH 7.4). A vacuum was applied for 2 h during a total 48-h incubation. Fixed nodules were embedded in plastic (Technovit 7100; Heraeus-Kulzer) according to the manufacturer’s recommendations. Sections (5 µm) were made using an RJ2035 microtome (Leica Microsystems). Sections were stained using 0.05% (w/v) Toluidine Blue O. Images were taken with a DM5500B microscope equipped with a DFC425c camera (Leica Microsystems).
All nodules were fixed in buffer containing 4% (w/v) paraformaldehyde mixed with 3% (v/v) glutaraldehyde in 50 m
7
Microscopic Analysis of Peanut Root Nodules
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For a macroscopic view, pictures were taken under a Nikon SMZ25 stereomicroscope and images were processed using NIS Elements AR 5.10.00. For semithin sectioning, peanut root nodules were fixed at 4°C overnight with 3% paraformaldehyde (wt/vol) and 2.5% glutaraldehyde (vol/vol) in 0.05 M sodium cacodylate buffer (pH 7.2). The fixed samples were dehydrated in ethanol gradient and subsequently embedded in LR white (Electron Microscopy Science), and semithin sections were generated according to the manufacturer's protocol. Sections (1 μm) were cut with an RJ2035 microtome (Leica Microsystems). For Toluidine Blue staining, the sections were stained for 5 min in 0.05% Toluidine Blue O (Sigma) and analyzed using a DM5500B microscope equipped with a DFC425C camera (Leica Microsystems). For confocal microscopy, sections were cut using a vibratome (Leica Microsystems, VT-1000S) and stained with Calcofluor white (Sigma) and SYTO13 (Life Technologies). Confocal images were captured with a TCS-SP8 (Leica) using excitation (ex) of 488 nm and emission (em) of 500 to 530 nm for SYTO13 and 405 nm ex and 410 to 430 nm em for Calcofluor white. Images were processed with LAS AF Lite version software and prepared for presentation with Photoshop CS6 (Adobe Systems Inc.).
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