Nine to ten week old male C57BL/6 J mice were fed either a high-fat diet (HF) containing 24 % (wt/wt) fat (21.3 % lard and 2.3 % soy oil, n = 9), HF diet supplemented with FO (EPAX 6000 TG®, a generous gift of Epax A/S, Ålesund, Norway) (15.7 % lard, 2.3 % soy oil and 5.8 % FO, n = 6) or the HF diet supplemented with KO (Superba™, a generous gift of Aker BioMarine Antarctic AS, Oslo, Norway) (15.7 % lard, 2.3 % soy oil and 5.7 % KO, n = 6) and water ad libitum for 6 weeks. Diets were packaged in airtight bags and freeze stored until use to prevent lipid oxidation. Mice were housed in groups of three per cage at a constant temperature of 22 ± 2 °C and a light/dark cycle of 12/12 h. Body weights of the animals were measured approximately every seventh day and food intake was measured three times in the beginning of the 6-week study to optimize the food supply. At the end of study animals were fasted overnight, anesthetized with 2 % isoflurane (Schering-Plough, UK) and blood was collected by heart puncture. After collection all tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until further analysis. The animal experiments were carried out with ethical permission obtained from the Norway State Board for Biological Experiments and followed the Norwegian Research Councils ethical guidelines.
Isoflurane
Manufactured by Merck & Co
Sourced in United Kingdom
Isoflurane is a volatile anesthetic agent used in various medical and laboratory settings. It is a clear, colorless, and nonflammable liquid with a characteristic odor. Isoflurane is primarily used for the induction and maintenance of general anesthesia in both human and veterinary medicine.
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Lab products found in correlation
Flunixin meglumine, by Merck & Co (1 mentions)
Urethane, by Merck Group (1 mentions)
Cp376395, by Bio-Techne (1 mentions)
Chol2, by Roche (1 mentions)
Cobas c111 system, by Roche (1 mentions)
Elisa, by Crystal Chem (1 mentions)
Temgesic, by Merck & Co (1 mentions)
λ carrageenan, by Merck Group (1 mentions)
Buprenorphine, by Merck & Co (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Beuthanasia d, by Merck & Co (1 mentions)
9 protocols using isoflurane
1
High-Fat Diet and Omega-3 Supplementation in Mice
2
Antagonist Receptor Binding and Anesthesia
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The selective CRF1 receptor antagonist CP376395 (Tocris, Westwoods Business Park, Ellisville, Missouri, USA), corticotropin-releasing factor (CRF) (Sigma-Aldrich, St. Louis, Missouri, USA), and urethane (Sigma-Aldrich) were dissolved in saline (0.9% NaCl). The veterinary pentabiotic (Fort Dodge, Campinas, SP, Brazil), isoflurane (Cristrália, Itapira, São Paulo, Brazil), and the nonsteroidal anti-inflammatory flunixin meglumine (Banamine, Schering-Plough, Cotia, SP, Brazil) were used as supplied by the manufacturers.
3
Plasma Lipid and Adipokine Analysis
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Animals were fasted for 6 hours, anesthetized with 2% isoflurane (Schering-Plough, Kent, UK) and blood was collected by heart puncture before the mouse was euthanized. The blood was centrifuged, EDTA-plasma separated and stored at −80 °C prior to further analysis. Liver, epididymal white adipose tissue (eWAT), inguinal WAT (iWAT), skeletal muscle (gastrocnemius) and BAT were collected and immediately frozen in liquid nitrogen and stored at −80 °C until further analysis.
Fasting EDTA-plasma samples at study endpoint were analyzed for Total Cholesterol, HDL Cholesterol, LDL Cholesterol, Triglycerides, Non-esterified fatty acids (NEFA) using the Cobas C111 System (Roche Diagnostics GmbH, Mannheim, Germany). Standard kits were used for all except for NEFAs that were analyzed using the NEFA FS kit (DiaSys, Diagnostic Systems GmbH, Germany). Lipids were measured by enzymatic colorimetric assays with specific reagents from Roche Diagnostics for total cholesterol (CHOL2, Cat. No. 04718917190), triglycerides (TRIGL, Cat. No. 04657594190), HDL cholesterol (HDL, Cat. No. 05401488190), and LDL cholesterol (LDL C, Cat. No. 04657578190). Insulin (Cat. No. 90080) and Leptin (Cat. No. 90030) were measured by ELISA from (Crystal Chem, Downers Grove, IL, USA).
Fasting EDTA-plasma samples at study endpoint were analyzed for Total Cholesterol, HDL Cholesterol, LDL Cholesterol, Triglycerides, Non-esterified fatty acids (NEFA) using the Cobas C111 System (Roche Diagnostics GmbH, Mannheim, Germany). Standard kits were used for all except for NEFAs that were analyzed using the NEFA FS kit (DiaSys, Diagnostic Systems GmbH, Germany). Lipids were measured by enzymatic colorimetric assays with specific reagents from Roche Diagnostics for total cholesterol (CHOL2, Cat. No. 04718917190), triglycerides (TRIGL, Cat. No. 04657594190), HDL cholesterol (HDL, Cat. No. 05401488190), and LDL cholesterol (LDL C, Cat. No. 04657578190). Insulin (Cat. No. 90080) and Leptin (Cat. No. 90030) were measured by ELISA from (Crystal Chem, Downers Grove, IL, USA).
4
Animal Anesthesia and Pain Management Protocol
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All animals had free access to standard food and water, and were housed in a room with a 12-hour dark-light cycle. Anesthesia was induced and maintained by inhalation of isoflurane (2–3% v/v, Schering-Plough, Stockholm, Sweden) mixed with air (∼1 L/min) in an isoflurane vaporizer (Ohmeda Isotec 5, Simtec engineering, Askim, Sweden). Temgesic® (0.1 ml/100 g body weight, Schering-Plough, Stockholm, Sweden) was given as a post-operative pain reliever in all small operative procedures. Gothenburg Ethical Board for Animal Experiments approved the study.
5
Islet Cell Transplantation in Mice
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Six to eight week old female recipient B6.ROSA-tomato mice were anaesthetised using inhalation anaesthesia (isoflurane; Schering-Plough, Kenilworth, NJ, USA) combined with analgesia using buprenorphine (0.15 mg/kg s.c.; RB Pharmaceuticals, Slough, UK). The islet suspension (30 μl) was injected between the capsule and renal parenchyma of the left kidney using a blunt cannula connected to a gastight syringe (Hamilton, Reno, NV, USA), as described previously [30 (link)].
6
Evaluating Endoglin Modulation in Inflammation
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λ-Carrageenan (catalog #2329535), lipopolysaccharide (LPS) (catalog #L3129) and Fluorescein Isotiocyanate Dextran (FITC-Dextran) (catalog #FD40) were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant human endoglin (rhEndoglin; Eng) (Catalog #1097-EN-025) was purchased from R&D Systems (Minneapolis, MN, USA). Isoflurane and buprenorphine (Schering-Plough; Madrid, Spain) were used for anesthesia and analgesia respectively.
7
Tissue Collection and Plasma Extraction
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After four weeks of diet treatment, fasted rats were anaesthetized by inhalation of 2% isoflurane (Schering-Plough, Kent, UK). The abdomen was opened in the midline and blood was drawn by cardiac puncture in Vacutainer tubes containing 7.5% ethylenediaminetetraacetic acid (EDTA) and immediately chilled on ice for a minimum of 15 min. The samples were centrifuged and plasma was stored at −80 °C prior to analysis. Heart, liver, and adipose tissues (mesenteric, epididymal, perirenal, and subcutaneous white adipose tissue depots) were collected and weighed. A sample from each liver was removed for β-oxidation analysis, while the remaining parts of the liver and the other tissues were immediately snap-frozen in liquid nitrogen and stored at −80 °C until further analysis.
8
Murine Skin Wound Healing with Tryptophan
9
Olfactory Bulbectomy Model in Mice
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Animals used were 2-3 month old male C57BL/6J mice weighing 25-30 g. The OB and sham-operation (sham) procedure was performed as previously described (Linge et al., 2013; (link)2016) . Mice were anesthetized with isoflurane (2%; Schering Plough, United Kingdom) to perform the bilateral olfactory bulbectomy. In brief, a midline sagital incision was made in the skin overlying the skull, and a burr hole was drilled through which both olfactory bulbs were bilaterally aspired using a suction pump. Finally, the hole was filled with bone wax to avoid bleeding. After bulbectomy/sham surgery, a four week period was waited for the animal recovery and the development of the OB syndrome before the initiation of drug treatment was confirmed by peripheral hyperactivity in the open field test. At the end of the study, animals were sacrificed and the lesions were verified to discard frontal pole lesions and/or incomplete removal of olfactory bulbs. Sham operations were done in the same way, although the bulbs were left intact.
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