Root tips (5 mm, 3–4 seedlings per replicate) were excised from the seedlings exposed to different treatments (22 h) and were frozen in liquid nitrogen. The frozen root tips were ground in liquid nitrogen, and a known weight (10–15 mg) of ground tissue was used for further extractions. In brief, the tissue was added to methanol, incubated at 25 °C for 10 min, and dried using a speedy vac. The pellets were washed in 70 mM lanthanum chloride (LaCl3) followed by precipitation using 1N potassium hydroxide (KOH). The supernatants were harvested and frozen until they were needed for further use. GABA concentrations were measured on an OMEGA plate-reading spectrophotometer following the GABase enzyme assay [4 (link)]. To determine the GABA efflux from oocytes, the oocytes that had been injected with TaALMT1, VvALMT9 cRNA, or water (controls) were imaged in groups of four to five using a stereo zoom microscope (SMZ800) with a Nikon (cDSS230) camera 48 h after injection. The oocytes were exposed to treatment solutions (10 min), as indicated in the figure legends, followed by harvesting of the treatment solutions and snap freezing in liquid nitrogen. All the samples were assayed for GABA efflux using the GABase enzyme assay as per published protocols [28 (link)].
C dss230
Manufactured by Nikon
Sourced in Japan
The C-DSS230 is a compact digital spectrophotometer from Nikon. It is designed for accurate and reliable measurement of various samples in laboratory settings. The device features a high-resolution display, intuitive user interface, and advanced optics for precise spectral analysis.
Automatically generated - may contain errors
Lab products found in correlation
C2 confocal microscope, by Nikon (3 mentions)
Ds u2, by Nikon (2 mentions)
H 7650 tem, by Hitachi (1 mentions)
Nis elements software, by Nikon (1 mentions)
Nis elements image acquisition software, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Femtojet microinjector, by Eppendorf (1 mentions)
12 protocols using c dss230
1
Quantification of GABA Efflux from Oocytes
2
Probing Myc-enok in Salivary Glands
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Transgenic flies carrying UAS-Myc-enok were crossed with sgs-GAL4 driver line. Salivary glands from third instar larvae were isolated and polytene chromosomes were stained with α-TRX and α-Myc using standard protocol [64 ].
For embryonic staining, stage 15 embryos were dechorionated and GFP-negative embryos were separated under an epifluorescent stereo microscope (Nikon, C-DSS230) and were stained using standard protocol [64 ]. All images were acquired using the Nikon C2 Confocal Microscope.
For embryonic staining, stage 15 embryos were dechorionated and GFP-negative embryos were separated under an epifluorescent stereo microscope (Nikon, C-DSS230) and were stained using standard protocol [64 ]. All images were acquired using the Nikon C2 Confocal Microscope.
3
Visualizing Lignin in Switchgrass Stems
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To visualize lignin content in switchgrass stem, switchgrass hand‐cut cross sections of the middle of 1NE3 stem were stained with 1% phloroglucinol/HCl for 1 min. Digital images were captured under a light microscope (Nikon, Shizuoka, Japan C‐DSS230) (Liu et al., 2020 (link)). The middle parts of 1NE3 stems were fixed in 2.5% glutaraldehyde. Stem epidermal cells were photographed under scanning electron microscope (SEM). The cell diameter of cross section cells (n = 80) and long cell length of epidermal cells (n = 50) were measured from SEM photographs using ImageJ software (https://imagej.en.softonic.com ). The cell number was calculated by the ratio of the internode length to the long cell length (n = 50).
4
Oocyte Water Permeability Measurement
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Water permeability was assessed by placing individual oocytes into small plates containing hypotonic ND48 medium (ND96/2). This was a two-step procedure in which we first equilibrated the internal osmolarity to isosmotic ND48 with mannitol (190 mOsm), and later placed the oocyte into ND48 (95 mOsm). Oocyte swelling was recorded by a Nikon CDSS230 stereomicroscope coupled to a Nikon DS-U2 camera. NIS Elements software was used to configure the imaging protocol for data acquisition. Oocyte volume was obtained from the oocyte section area calculated in ImageJ. Water permeability (Pf) was calculated from the initial rate of volume increase with the formula Pf = Vo[d(V/Vo)/dt]/[So x Vm (Osmin - Osmout)], where Vo is the initial oocyte volume (in cm3), So the initial oocyte area (in cm2), Vm the molar volume of water (18 cm3/mol), Osmin the internal oocyte osmolarity (190 mOsm), and Osmout the ND48 osmolarity.
5
Insect Tissue Ultrastructure Analysis
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After having been treated with dsGFP or dsCpr21L for 2 days, five nymphs of the 4th instar were sampled for each group and placed on ice, and their heads and thoraces were quickly removed with tweezers under a Nikon C-DSS230 stereomicroscope. The remaining part of the insect body without the head and thorax was quickly soaked in 2.5% glutaraldehyde solution and fixed overnight at 4 °C. After the samples were fixed well, subsequent processing was performed using a previously reported method [8 (link),23 (link)]. Semi-thin sections (2 μm) were cut using glass knives on an LKB Bromma 11,800 pyramitome (LKB, Bromma, Sweden) and stained with methylene blue. The ultra-thin sections were prepared with a diamond knife using PowerTome-PC (RMC, Boeckeler Instruments, Tucson, AZ, USA). The sections were stained with 3% uranyl acetate and alkaline lead citrate and were observed using a Hitachi H-7650 TEM (Hitachi, Tokyo, Japan).
6
Quantifying Shoot and Root Traits
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To measure the shoot fresh weight, seedlings were sectioned at the root-shoot junction, and twelve groups of excised shoots were immediately measured on an analytical balance (two shoots were used in each group). The number of emerged LRs of at least 20 seedlings was counted every day using a dissecting microscope (C-DSS230, Nikon, Japan) for 7 days. For RH measurements, digital images were obtained every day from the primary root segment for a total of 7 days, located 2 mm above the root tip, using a dissecting microscope and a magnification of 15×. Then RH length and density were quantified with ImageJ software (ImageJ 1.48u; Rasband, Bethesda, MD, United States). The number of emerged RH branches of at least 20 seedlings was counted by using digital images with a dissecting microscope and a magnification of 50×.
7
Imaging Whole SCG and Tissue Sections
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Bright-field images of whole SCGs and tissue sections stained by in situ hybridization or immunohistochemically were captured on a Nikon C-DSS230 dissecting microscope with an SMZ2800 objective using Nikon “digital sight” hardware and NIS-elements software.
8
Analyzing Abdominal Pigmentation in Male Flies
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For analyzing loss of abdominal pigmentation, male flies of the desired genotypes were transferred to 70% ethanol to dehydrate. The dehydrated flies were then dissected under Olympus SZ51 stereomicroscope on a dissection slide with the help of fine needle. The abdominal portion of the flies were isolated and rehydrated in water for 5 min. After rehydration, abdomens were dissected for cuticle preparation. A coverslip was placed over the cuticle and observed under Nikon C-DSS230 epifluorescent stereomicroscope at ×3.5 magnification using white light for imaging. For analyzing the immunostaining data, embryos were observed at ×20 using Nikon C2 Confocal Microscope. Images were acquired by exposure of embryos to lasers, i.e., DAPI (excitation 405 nm and emission 447 nm) and Cy3 (excitation 561 nm and emission 785 nm) in sequential manner. All polytene chromosomes were visualized at ×60 magnification using Nikon C2 Confocal Microscope. Images were acquired by exposing to DAPI (excitation 405 nm and emission 447 nm), Alexa 488 (excitation 488 nm and emission 525 nm), and Cy3 (excitation 561 nm and emission 785 nm) in a sequential manner. Nikon NIS Elements image acquisition software (Version: 5.21.00) was utilized for imaging and analysis.
9
Whole-Mount In Situ Hybridization in Zebrafish Embryos
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WISH of zebrafish embryos was performed as described previously44 (link) using probes of cd146, lyve1b, dll4, tbx20, ephrinB2, msr and flt4. The embryos were observed with a Nikon C-DSS230 stereo-microscope, and the images were taken with a Nikon DS-U2 camera using NIS-Elements (version F3.0).
10
Lignin Content Analysis Protocol
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To assess lignin content, we added 0.15 g DMS samples to 2 ml tubes and stained with 2 ml phloroglucinol/HCl (1/12, w/w) for 5 min. The hand-cut section of the middle of the first internode from top of E3 stage tiller was stained with phloroglucinol/HCl for 1 min. Photos were taken with a digital camera on a microscope (Nikon, C-DSS230).
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