MSU were purchased from the Jiancheng Institute of Biotechnology (Nanjing, China). Pentobarbital sodium, paraformaldehyde and physiologic saline solution were purchased from Shanghai Chemical Reagent Co. (Shanghai, China). The darutoside with more than 97.4% purity (batch number: BCBS7438) were obtained from Chengdu Cloma Biological Co., Ltd. (China) and the HPLC chromatography was shown in Supplementary Figure S1 . Enzyme-linked immunosorbent assay (ELISA) kits for interleukin-8 (IL-8), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) assay were purchased from Assay R&D Systems. Nuclear factor kappa-B (NF-κB) and interleukin-10 (IL-10) were purchased from Biowest (Nuaillé, France) and HyClone (Logan, Utah, United States), respectively. Blood uric acid (UA) was bought from Chondex (Redmond, WA, United States). LC-MS grade acetonitrile (ACN) and methanol (MeOH) were purchased from Merck (Darmstadt, Germany), and formic acid (FA) was purchased from Sigma Chemicals Ltd. (St. Louis, MO, United States). Double-distilled water was purified using a Millipore water purification system (Millipore, Bedford, MA). Leucine enkephalin as a standard substance with the purity more than 99.2% were obtained from Hyclone (Logan, UT, United States). Other reagents used were of analytical grade and were purchased locally.
Sodium pentobarbital
Manufactured by Shanghai Chemical Reagent
Sourced in China
Sodium pentobarbital is a chemical reagent commonly used in research and analytical laboratories. It is a barbiturate compound that can be used as a sedative and hypnotic agent. The core function of sodium pentobarbital is to provide a controlled, consistent, and reliable substance for various applications within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Lc ms grade acetonitrile acn, by Merck Group (1 mentions)
Methanol meoh, by Merck Group (1 mentions)
Millipore water purification system, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Shanghai Chemical Reagent (1 mentions)
Xylene, by Guangzhou Chemical (1 mentions)
Formaldehyde, by Guangzhou Chemical (1 mentions)
Anhydrous ethanol, by Guangzhou Chemical (1 mentions)
Methanol, by Tedia (1 mentions)
C fos, by Abcam (1 mentions)
Anti pten antibody, by Abcam (1 mentions)
Anti vegfa antibody, by Abcam (1 mentions)
Anti hif 1 alpha antibody, by Abcam (1 mentions)
Anti akt1 phospho s473 antibody, by Abcam (1 mentions)
Ab32199, by Abcam (1 mentions)
L nitroarginine methyl ester l name, by Merck Group (1 mentions)
Ab1316, by Abcam (1 mentions)
Freund s complete adjuvant fca, by Merck Group (1 mentions)
11 protocols using sodium pentobarbital
1
Darutoside Modulates Inflammatory Responses
2
Establishing Rabbit VX2 Liver Tumor Model
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The study was conducted in strict accordance with the "Guide for the Care and Use of Laboratory Animals," 8 th edition (International Publication No: 978-0-309-15400-0) and approved by the Institutional Animal Care and Use Committee of the China Medical University (CMU) Animal Care and Use Committee.
Adult male New Zealand white rabbits were purchased from the experimental animal center of the CMU. Thirtyeight rabbits (weight: 2.5-3 kg, 2.7 ± 0.2 kg, aged 5 months) were used to establish the rabbit VX2 liver tumor model. In all the experiments, animals were anesthetized by intramuscular injection of pentobarbital sodium (25 mg/kg) (Shanghai Chemical Reagent Co., Shanghai, China). The VX2 liver tumors were implanted as previously described.
[ 16 ] The VX2 tumors were injected into the hind limb of a donor rabbit. The tumors were harvested when they reached 1 cm in diameter [Figure 1a]. The excised fresh tumor samples were cut into small cubes (~1 mm 3 ). The tumor fragments were then pushed into the liver lobe, percutaneously, through an 18-G needle under ultrasound guidance [Figure 1b]. Typically, the tumors were suitable for experiment within 2 weeks of implantation.
[ 17 ] Finally, the tumors were observed using a clinical 3.0-T magnetic resonance imaging (MRI) scanner (GE Healthcare, Milwaukee, WI, USA).
Adult male New Zealand white rabbits were purchased from the experimental animal center of the CMU. Thirtyeight rabbits (weight: 2.5-3 kg, 2.7 ± 0.2 kg, aged 5 months) were used to establish the rabbit VX2 liver tumor model. In all the experiments, animals were anesthetized by intramuscular injection of pentobarbital sodium (25 mg/kg) (Shanghai Chemical Reagent Co., Shanghai, China). The VX2 liver tumors were implanted as previously described.
[ 16 ] The VX2 tumors were injected into the hind limb of a donor rabbit. The tumors were harvested when they reached 1 cm in diameter [Figure 1a]. The excised fresh tumor samples were cut into small cubes (~1 mm 3 ). The tumor fragments were then pushed into the liver lobe, percutaneously, through an 18-G needle under ultrasound guidance [Figure 1b]. Typically, the tumors were suitable for experiment within 2 weeks of implantation.
[ 17 ] Finally, the tumors were observed using a clinical 3.0-T magnetic resonance imaging (MRI) scanner (GE Healthcare, Milwaukee, WI, USA).
3
Nissl Staining of Rat Hippocampus
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Rats were deeply anesthetized with pentobarbital sodium (Shanghai Chemical Reagent Company) at the dose of 50 mg/kg and perfused transcardially with 0.9% NaClfor 5 min. Rats were then perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) for 20 min. Brains were removed, fixed in 4% PFA for 7 days at room temperature and embedded in paraffin. Following this, 3 µM-thick paraffin sections were excised from the dentate gyrus (DG) of the hippocampus and Nissl staining (56˚C for 30 min) was successfully optimized onto paraffin sections. Morphological changes of neurons were detected using a light microscope (magnifications, x40, x100 and x200).
4
Dosage-dependent Effects of Zoledronic Acid
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The following drugs and reagents were used in the present study: Injection of ZOL (Novartis International AG, Basel, Switzerland), injection of sumianxin II (Veterinary Institute of Medical Science Military Academy, Changchun, China), pentobarbital sodium (Shanghai Chemical Reagent Factory, Shanghai, China), toluidine blue (Amresco, Inc., Framingham, MA, USA), eosin hematoxylin, formaldehyde, xylene, anhydrous ethanol (all from Guangzhou Chemical Reagent Factory, Guangzhou, China) and chloral hydrate (Dalian Meilun factory, Dalian, China).
The dose of ZOL administered to the animals was calculated by the conversion formula of surface area based on that for humans. It was defined by the following equation: Dose for rabbits (2.0 kg)=[dose for humans (60 kg)x0.07]2/3=(5,000×0.07)2/3=50 µg. Therefore, a high dose of ZOL (ZH), a medium dose of ZOL (ZM) and a low dose of ZOL (ZL) group were established, which were administered ZOL at doses of 250, 50 and 10 µg/kg, respectively.
The dose of ZOL administered to the animals was calculated by the conversion formula of surface area based on that for humans. It was defined by the following equation: Dose for rabbits (2.0 kg)=[dose for humans (60 kg)x0.07]2/3=(5,000×0.07)2/3=50 µg. Therefore, a high dose of ZOL (ZH), a medium dose of ZOL (ZM) and a low dose of ZOL (ZL) group were established, which were administered ZOL at doses of 250, 50 and 10 µg/kg, respectively.
5
Extraction and Analysis of Barbital Derivatives
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Barbital was obtained from Shenyang Trial Three Biochemical Technology Development Co., Ltd. (Shenyang, China). Phenobarbital and pentobarbital sodium were purchased from Shanghai Chemical Reagent Factory (Shanghai, China). Formic acid, acetic acid, 2-octanone, 2-nonanone, 2-undecanone, 1-octanol, dihexyl ether (DHE), trifluoroacetic acid (TFA), and diclofenac sodium were all purchased from Aladdin Chemical Reagent Co. (Shanghai, China). Hydrochloric acid (HCl), sodium hydroxide (NaOH), potassium hydroxide (KOH), and trisodium phosphate (Na3PO4) were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methanol was from Tedia (Fairfield, OH, USA). Formic acid, Methanol, and acetic acid were of chromatographic purity grade while other chemicals were of analytical grade. Milli-Q water purification system (Mollsheim, France) was employed to produce deionized water.
Accurel PP 1E (R/P) flat membrane (polypropylene membrane, average thickness of 100 μm) was from Membrana (Wuppertal, Germany). The standard 1000 μL pipette tips were from Kirgen (Shanghai, China). The Eppendorf safe lock 2.0 mL PP tubes were obtained from Eppendorf AG (Hamburg, Germany).
Accurel PP 1E (R/P) flat membrane (polypropylene membrane, average thickness of 100 μm) was from Membrana (Wuppertal, Germany). The standard 1000 μL pipette tips were from Kirgen (Shanghai, China). The Eppendorf safe lock 2.0 mL PP tubes were obtained from Eppendorf AG (Hamburg, Germany).
6
Gallbladder Duct Ligation in Rabbits
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A total of 32 New Zealand rabbits of either sex (11 males, 21 females), weighing 2.6 kg (range, 2-3 kg) at the beginning of the study, were obtained from Guangdong Medical Laboratory Animal Center, China (order no. 44411600002998). Rabbits were housed in separate cages in the animal experimental center, with an ambient temperature of 18-24˚C and a 12 h light/dark cycle. Rabbits were provided ad libitum access to food and water during the experimental period. Rabbits were anesthetized by injection of 30 mg/kg sodium pentobarbital in the ear vein (Shanghai Chemical Reagent Company, China National Pharmaceutical Group Corporation) and intramuscular injection of 5 mg/kg xylazine hydrochloride (Shanghai Chemical Reagent Company, China National Pharmaceutical Group Corporation) after 12 h of fasting. By means of surgical laparotomy, the cholecystic duct was ligated with a 3/0 surgical suture, whereas cystic vessels were retained to ensure good blood supply and to avoid ischemic necrosis (Fig. 1 ). Direct gallbladder puncture was performed as far as possible from gallbladder vessels using a 22-gauge butterfly needle. Bile was aspirated, and the gallbladder was irrigated 3-4 times with saline until clear. The surgical time ran between 45 and 60 min. Anesthesia with a standard dose was well tolerated.
7
Syk/Ras/c-Fos Pathway Inhibitor R406 Protocol
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ADR was obtained from Hisun Pfizer Pharmaceuticals Ltd. (cat. no. 15029611; Zhejiang, China). Sodium pentobarbital was obtained from Shanghai Chemical Reagent Company (cat. no. 127K1005; Shanghai, China). Total RNA from renal cortex tissues was extracted by TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. Antibodies against phosphorylated (p)-Syk, Ras, p-MAPK extracellular signal regulated kinase (ERK; MEK)1/2, p-ERK1/2, c-Fos and β-actin were purchased from Abcam (Shanghai, China; cat. nos. ab79193, ab16907, ab194754, ab76299, ab209794, ab8226). The Syk/Ras/c-Fos pathway inhibitor R406 (inhibitor of Syk) was purchased from AbMole BioScience, (Shanghai, China). All the materials under current study were non-toxic to animals and cell cultures, including all biological and synthetical agents used for immunopharmacological studies.
8
Hippocampus Histopathology in Mice
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Four mice from each group were selected for hippocampus histopathology. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (2 mL/kg; Shanghai Chemical Reagent Company) after fasting for 12 h and taken bilateral hippocampus tissues, fixed with 4% paraformaldehyde, dehydrated in ethanol and xylene, embedded in clear paraffin, sectioned, delayed, stained with hematoxylin–eosin, further dehydrated and sealed, and observed under a light microscope.
9
Porcine Anesthesia and Euthanasia
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The pigs were sedated with 25 mg/kg sodium pentobarbital (concentration, 2%) (Shanghai Chemical Reagent Company, China) via marginal ear vein injection. The flank and back hairs were trimmed with electric clippers and the skin was scrubbed with soap and water. While still sedated, the pigs were killed by an intracardiac injection of pentobarbital and dissected away the skin.
10
Molecular Mechanisms of HIF-1α Regulation
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L-Nitro-arginine-methyl-ester (L-NAME) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany; batch no. N5751-10), and sodium pentobarbital was purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Anti-HIF-1 alpha antibody, anti-PTEN antibody, anti-VEGFA antibody, anti-PI3K p85 antibody, anti-PI3K p85 (phospho Y607) antibody, and anti-AKT1 (phospho S473) antibody were purchased from Abcam (batch no. Ab1; ab32199; ab1316; ab86714; ab182651; ab81283; dilution 1 : 200; 1 : 100; 1 : 100; 1 : 100; 1 : 200; 1 : 200); TRIB3 polyclonal antibody, AKT (L321) polyclonal antibody, and AKT (phospho-T308) polyclonal antibody were purchased from Bioworld (batch no. BS60451; BS1502; BS4647; dilution 1 : 100; 1 : 100; 1 : 100).
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