The herbicide susceptibility of genome-edited lines was measured within test tubes (diameter, 2.5 cm; height, 15 cm) containing 10 ml of a Murashige–Skoog (MS) solid medium with herbicide. Five dehusked mature seeds of homozygous his1 T3 or wild-type (control) rice plants were surface sterilized via two treatments with 4% sodium hypochlorite for 20 min followed by five rinses with sterilized water. The seeds were then immersed in sterilized water for 2 days at 30°C, after which germinated seeds were transferred to tubes containing the solid MS medium composed of half-strength MS salts and agar (1 g/L) containing herbicide; BBC 0.1 µM, BBC-OH 0.1 µM, and 0.3 µM; Mesotrione (MST); Sulcotrione (SLT); Tembotrione (TMT) 0.05 µM and 0.1 µM; and Tefuryltrione (TFT) 0.1 µM and 0.3 µM, cultured at 27°C for 7 to 14 days with 16 h of light (40 μmol m−2 s−1) daily. BBC and BBC-OH were obtained from SDS Biotech (Tokyo, Japan), while MST, SLT, TMT, and TFT were obtained from Fujifilm Wako (Tokyo, Japan).
Tandem mass tag (tmt)
Manufactured by Fujifilm
Sourced in Japan
The TMT (Tandem Mass Tag) is a laboratory instrument designed for protein quantification and identification. It utilizes mass spectrometry technology to enable the simultaneous analysis of multiple samples, allowing for comparative protein expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Specific pathogen free male c57bl 6 mice, by Orient Bio (1 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions)
Tandem mass tag (tmt), by Thermo Fisher Scientific (1 mentions)
L3000, by Rigol (1 mentions)
β catenin, by Merck Group (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg h l antibody, by Thermo Fisher Scientific (1 mentions)
4 protocols using tandem mass tag (tmt)
1
Herbicide Susceptibility of Rice Mutants
2
TMT-Induced Seizures in Mouse Hippocampi
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Specific pathogen-free male C57BL/6 mice (of age 8 weeks and weight 20–25 g) were purchased from Orient Bio, Inc. (Seoul, Korea) and used after a 2-week period of quarantine and acclimatization. All experiments and protocols used in this study were approved by the Institutional Animal Care and Use Committee of Chonnam National University (CNU IACUC-YB-2018-69), and the animals were cared for in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
TMT (Wako, Osaka, Japan) was dissolved in sterilized 0.9% saline. To assess the effects of TMT-induced seizures in mouse hippocampi, mice were sacrificed, and the brains were dissected 3 days after a single intraperitoneal (ip) injection of vehicle (0.9% saline) or 2.6 mg/kg of TMT solution (n = 6 mice per group), as adapted from a previously described protocol [13 (link)]. All the animals were euthanized by decapitation after 4 days after TMT treatment.
TMT (Wako, Osaka, Japan) was dissolved in sterilized 0.9% saline. To assess the effects of TMT-induced seizures in mouse hippocampi, mice were sacrificed, and the brains were dissected 3 days after a single intraperitoneal (ip) injection of vehicle (0.9% saline) or 2.6 mg/kg of TMT solution (n = 6 mice per group), as adapted from a previously described protocol [13 (link)]. All the animals were euthanized by decapitation after 4 days after TMT treatment.
3
Comparative Proteomic Analysis of Samples
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The cells were collected, and the experiments were carried out using TMT (Thermo Fisher) approach in triplicates for each group. The total proteins were extracted and enzymatic desalination using XBridge peptide BEH Column 18 (5 µm, Waters Corporation, Framingham, USA). TMT reagents were added to each sample, and ammonia (Wako Pure Chemical Industries Ltd., Osaka, Japan) was added to terminate the reactions. The samples were then lyophilized overnight and subject to liquid chromatography‐tandem mass spectrometer (LC‐MS/MS, L‐3000, Rigol, Beijing, China). Quantification and normalization of protein spots were performed using Proteome Discoverer 2.4 (Thermo Fisher) and Homo Sapient Swiss‐Prot database. The GO was performed, and the identified proteins of each group of samples in the experiment were uploaded to the KEGG (http://www.genome.jp/kegg ) website, and the map results of all pathways were obtained.
4
Immunofluorescent Staining of Neural Markers
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Anti-goat IgG antibody conjugated to fluorescein isothiocyanate was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Rabbit polyclonal antibodies against ionized calcium-binding adapter molecule 1 (Iba1; Wako Pure Chemical Industries, Ltd., Osaka, Japan) and β-catenin (Sigma-Aldrich Co., St. Louis, MO, USA), goat polyclonal antibody against doublecortin (DCX; Santa Cruz Biotecchnology, Santa Cruz, CA), rat monoclonal antibody against 5-bromo-2′-deoxyuridine (BrdU; Abcam, Ltd., Cambridge, MA, UK), and mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFAP; Sigma-Aldrich Co., St. Louis, MO, USA), nestin (Millipore Co., Boston, MA, USA), and neuronal nuclear antigen (NeuN; Chemicon International, Temecula, CA, USA) were used as primary antibodies. Alexa-Fluor 594-conjugated anti-rat IgG (H+L) antibody, Alexa-Fluor 488-conjugated anti-rabbit IgG (H+L) antibody, and Alexa-Fluor 488-conjugated anti-mouse IgG (H+L) antibody were obtained from Molecular Probes (Eugene, OR, USA). Lithium carbonate and TMT were supplied by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals used were of the highest purity commercially available.
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