Animals (n = 4–5 per group) were deeply anaesthetized with sodium pentobarbital (20%; Eutasil; CEVA) and transcardially perfused with 0.9% saline solution. Brains were removed and post‐fixed in Golgi‐Cox solution for 2 weeks and then transferred to 30% sucrose solution for further processing, as previously described.27 Dendritic length and neuronal branching from dorsal and ventral hippocampal DG and basolateral amygdala (BLA) were assessed. For each animal, 7–10 neurons fulfilling previously described criteria were analysed per subregion.28 Dendritic branches were reconstructed at ×1,000 magnification on a motorized microscope (Axioplan 2; Carl Zeiss; for BLA samples and Olympus BX‐53; Olympus; for the remaining samples) and Neurolucida software (MBF Bioscience). Three‐dimensional analysis of the reconstructed neurons was performed using the NeuroExplorer software (MBF Bioscience). The number of dendritic branching was evaluated in 3D Sholl analysis, measuring the number of concentric radial intersections at intervals of 20 µm. Measures from individual neurons were averaged per animal and compared among experimental groups.
Eutasil
Manufactured by Ceva
Sourced in France, Portugal
Eutasil is a laboratory equipment product designed for general laboratory use. It serves as a versatile tool for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Neuroexplorer software, by MBF Biosciences (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Neurolucida software, by MBF Biosciences (1 mentions)
Rab0274, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by ITW Reagents (1 mentions)
Hematoxylin and eosin h e, by Merck Group (1 mentions)
Biodec r, by Bio-Optica (1 mentions)
Rompun, by Bayer (1 mentions)
Mowiol mounting medium, by Merck Group (1 mentions)
P6148, by Merck Group (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Cm3050, by Leica (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Pimonidazole hydrochloride, by Hypoxyprobe (1 mentions)
10 protocols using eutasil
1
Dendritic Complexity in Hippocampus and Amygdala
2
Survival Analysis of Experimental Treatment
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The defined end-point for the survival analysis was a 20% loss of body weight plus oedema/ascites and decreased movement. When this endpoint was reached, animals were euthanized with an anaesthetic overdose (sodium pentobarbital 100 mg/kg, Eutasil, CEVA). Only those animals reaching the end-point before evaluation at study-end were considered valid and used in the survival analysis.
3
Mouse Plasma IL-1β Quantification
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Blood was collected from the animals under anesthesia by cardiac puncture using a heparinized tube and syringe and centrifuged for 5 min at 11,000× g to separate the plasma. The plasma was then stored at −20 °C for further analysis. Subsequently, the levels of IL-1β in the plasma of the mice were determined using the enzyme-linked immunosorbent assay (ELISA), according to the instructions of the kit (RAB0274, Sigma-Aldrich, St. Louis, MO, USA). Finally, the mice were euthanized with an injection of sodium pentobarbital (Eutasil, 200 mg/mL; Ceva, Portugal) 120 mg/kg.
4
Brain Tissue Collection and Biochemical Analysis
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Animals (n = 5–7 per group) were deeply anaesthetized with sodium pentobarbital (20%; Eutasil; CEVA, France) and transcardially perfused with cold 0.9% NaCl. Brains were rapidly collected, and brain regions were macrodissected and immediately stored at –80°C. Biochemical fractionation and immunoblotting of dorsal and ventral hippocampus were analysed, as described in the Appendix S1 .
5
Histopathological Evaluation of Knee Joints
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At the end of all experiments, a lethal dose of pentobarbitone (100 mg/kg, Eutasil®, CEVA, Algés, Portugal) was administered to the animals and perfused with 300 mL of a 4% (m/v) paraformaldehyde solution (PFA, Panreac, Barcelona, Spain) in 0.1 M PBS (phosphate buffered saline) (pH 7.4). The right knees were then excised and fixed in PFA 4% (v/v) for one week. Then, they were decalcified in a decalcifying solution (BiodecR, Bio-Optica, Milan Italy) for another week. The tissues were individually embedded in paraffin, and 4-µm serial sections were obtained at the medial levels in the sagittal plane. Lastly, the sections were stained with hematoxylin and eosin (HE) (Sigma-Aldrich, St. Louis, MO, USA) and examined under the microscope to check for any histopathologic abnormalities.
6
Euthanasia and Necropsy Protocol
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Animals from both acute and chronic toxicity experiments were sacrificed on the 29th day of the study by an intraperitoneal injection of ketamine (Eutasil 200 mg/mL, Ceva, Algés, Portugal) and xylazine (Rompun® 20 mg/mL, Bayer Healthcare S.A., Kiel, Germany), followed by exsanguination by cardiac puncture, as recommended by FELASA (Federation for Laboratory Animal Science Associations) [44 ]. A complete necropsy was performed, and the internal organs (heart, lungs, kidneys, spleen, and liver) were collected and weighed individually using a precision scale (KERN® PLJ 750-3N, Kern & Sohn GmbH, Balingen, Germany).
7
Histological Validation of Optogenetic Manipulations
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Histological analysis were performed after photostimulation experiments to confirm viral expression of ChR2-YFP and optical fiber placement. Mice were deeply anesthetized with pentobarbital (Eutasil, CEVA Sante Animale, Libourne, France) and perfused transcardially with 4% paraformaldehyde (P6148, Sigma-Aldrich). The brain was removed from the skull, stored in 4% paraformaldehyde overnight and kept in cryoprotectant solution (PBS in 30% sucrose) for one week. Sagittal sections (50 μm) were cut in a cryostat (CM3050S, Leica, Germany), mounted on glass slides with mowiol mounting medium (81381, Sigma-Aldrich, St. Louis, MO). Scanning images for YFP, RFP and transmitted light were acquired with an upright fluorescence microscope (Axio Imager M2, Zeiss, Oberkochen, Germany) equipped with a digital CCD camera (AxioCam MRm, Zeiss) with a 5X or 10X objective.
8
Quantifying Bone Marrow Hypoxia
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Three days after the hypoxic period, the subjects were intravenously injected (via tail vein) with 60-mg/kg pimonidazole hydrochloride (Hypoxyprobe, Inc, Burlington, USA), a misonidazole-based compound, which forms adducts with thiol groups of proteins, peptides, and amino acids specifically in hypoxic cells (pO2 < 10 mmHg) (81). Two hours later, rats were euthanized using 60-mg/kg sodium pentobarbital IV (Eutasil, Ceva Santé Animale, Libourne, France) and transcardially perfused with PBS. Quantification of BM hypoxic areas was performed using the ImageJ software in 10 high-power fields (×400 magnification) per animal.
9
Colorectal Cancer Development Protocol
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The animals from two groups were euthanized 11 weeks after the first DMH or EDTA–saline administration (groups CRC1 (n = 9) and CTRL1 (n = 6)), and the other animals from the remaining groups were sacrificed 17 weeks after the first administration (groups CRC2 (n = 8) and CTRL2 (n = 6); Figure 1 ). The animals were sacrificed in two timepoints in order to identify preneoplastic and neoplastic lesions, allowing a follow-up of the CRC development. All animals were sacrificed by an intraperitoneal overdose administration of sodium pentobarbital (Eutasil, CEVA, Libourne, France), followed by exsanguination via cardiac puncture, as indicated by the guidelines of the Federation for Laboratory Animal Science Associations (FELASA). A complete necropsy was performed in each animal. All organs were collected and weighed. The intestines were individualized and weighed separately. The colon was opened longitudinally, gently rinsed with saline in order to remove residual bowel contents and fixed flat in 10% buffered formalin.
10
Brain Tissue Fixation and Sectioning
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After behavioral assessment (14 weeks after surgery), animals were deeply anesthetized with sodium pentobarbital/Eutasil® (60 mg/kg; Ceva Santé Animale SA., Libourne, France) by intraperitoneal injection and transcardially perfused using 0.9% NaCl followed by 4% paraformaldehyde (PFA; Merck & Co. Inc., Kenilworth, NJ, USA) dissolved in 0.01 M phosphate-buffered saline (PBS). Rat brains were dissected and post-fixed in 4% PFA dissolved in 0.01 M PBS for 48 h, followed by immersion in a 30% sucrose solution dissolved in 0.01 M PBS. After sinking, the brains were sectioned into coronal slices (50 μm thick) using a vibratome (Leica Vibratome VT1000S; Leica Biosystems, Nußloch, Baden-Württemberg, Germany) and processed as free-floating sections.
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