Blood was collected from the submandibular vein into BD microtainer MAP microtubes coated with ethylene diamine tetraacetic acid (EDTA). Mice were then injected with Euthasol (0.002 mL/g; Patterson veterinary, code: 07–805-9296). A broncho-alveolar lavage (BAL) and left lung lobectomies were done to collect specimens for protein analysis. BAL fluid was collected via standard methods. Briefly, a midline incision at the neck was made and the trachea isolated by blunt dissection. The trachea was cannulated using a 22-gauge catheter that was secured in place using cotton suture. 1.5 mL of saline was delivered to the lungs via syringe and recovered via aspiration, this was repeated for collection of the total 1.5 mL volume administered. The left lobes of the lung were collected and flash frozen in liquid nitrogen for protein analysis. Mice were then transcardially perfused with phosphate-buffered saline (PBS) flush followed by 4% paraformaldehyde for 5 minutes. The right lobes of the lung were collected for histological analysis.
Euthasol
Manufactured by Performance Health
Sourced in United States
Euthasol is a veterinary euthanasia solution formulated for the humane and painless euthanasia of animals. It contains a combination of pentobarbital sodium and phenytoin sodium as the active ingredients.
Automatically generated - may contain errors
Lab products found in correlation
Microtainer map microtube, by BD (2 mentions)
Gentlemacs c tube, by Miltenyi Biotec (1 mentions)
Trizol reagent, by Zymo Research (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Serum separator tube, by BD (1 mentions)
Vibrating microtome, by Leica (1 mentions)
Sodium pentobarbital, by Performance Health (1 mentions)
Polyvinylpyrrolidone pvp 40, by Merck Group (1 mentions)
11 protocols using euthasol
1
Murine Lung Tissue and Fluid Collection
2
Tissue Collection and Processing for Hypothalamus Analysis
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Tissue was collected as previously described (Foradori et al. 2006 (link)). Briefly, at the end of the experiment (Week 13), all wethers were heparinized (20,000 U, intravenous) and killed with an intravenous overdose of sodium pentobarbital (Euthasol; Patterson Veterinary, Greeley, CO). Heads were removed and perfused via the carotid arteries with four liters of 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) containing 0.1% sodium nitrite. Blocks of tissue containing the hypothalamus were removed and stored in 4% PFA for 24 h at 4°C and transferred to a 20% sucrose solution until sectioning. Frozen coronal sections were cut at 50 µm with a freezing microtome into five parallel series and stored in cryopreservative solution until used for RNAscope and immunohistochemistry.
3
Pulsatile GH Secretion Dynamics Post-Injury
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Blood was collected via cardiac puncture at predetermined time points post injury (1, 7, 18, 25, 43 DPI; Fig. 1 ). GH secretion occurs in a pulsatile fashion and is influenced by the circadian rhythm, so all blood samples were collected at approximately the same time of day (zeitgeber 3–5; 09:00–11:00 h). Brains were collected from a subset of rats at 1, 7, and 43 DPI. Rats were injected (intraperitoneally) with Euthasol (0.002 mL/g, Patterson Veterinary, Greeley, CO, USA), and approximately 300 µL of blood were collected via cardiac blood draw using a needle and syringe coated in EDTA. Blood was transferred to an EDTA-coated tube and was centrifuged to collect plasma for each rat. Plasma samples were stored at −20°C until GH quantification. Immediately following blood collection, rats underwent transcardial perfusion with ice-cold 1× PBS followed by 4% paraformaldehyde (PFA). Brains were removed from the skull and drop fixed in 4% PFA for 24 h. For cryoprotection, brains were successive incubated in 15% and 30% sucrose, each for 24 h. Brains were frozen and cryosectioned in the coronal plane at 40 µm and mounted on superfrost slides and stored at −80°C.
4
Comprehensive Mouse Tissue Harvesting
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On the day of euthanasia, mice were administered an intraperitoneal injection (390 mg/kg) of Euthasol (pentobarbital) (Patterson Veterinary 07-249 805-9296) diluted in 0.9% sterile NaCl. Once mice were unresponsive, blood was collected via cardiac puncture in serum separator tubes (BD, 365,967). To separate the serum, each tube was centrifuged for 5 min at 15,000 × g. Mice were dissected to collect the lung and brain tissues as well as nasal wash fluid for downstream analysis. Nasal wash fluid was collected by pushing 1mL sterile PBS by catheter through the nasal pharynx. Right lobes of the lung were homogenized in 1mL sterile PBS using gentleMACS C tubes (Miltenyi Biotec, 130-096-334) and the m_lung_02 program on the gentleMACS Dissociator (Miltenyi Biotec, 130-093-235). Whole brains were homogenized similarly. For RNA analysis 300μL of lung homogenate, or 500μL or brain homogenate was added to 1mL TRIzol Reagent (Zymo R2050-1). For nasal wash RNA analysis, 500μL of nasal wash was added to 500μL of TRIzol Reagent. For cytokine analysis, 300μL of lung homogenate was centrifuged at 15,0000 × g for 5 min and the supernatant was collected.
5
Patch Clamp Recordings of Adolescent Rat mPFC
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All patch clamp recordings were done at the conclusion of behavioral testing in adolescence (Fig. 1 ). Rats were anesthetized with Euthasol (100 mg/kg, Patterson Veterinary) and then transcardially perfused with 60 ml of oxygenated slicing artificial CSF (aCSF) containing 34 mm sucrose, 11 mm glucose, 24 mm NaHCO3, 2.5 mm KCl, 1.25 mm NaH2PO4, 10 mm MgSO4, and 0.5 mm CaCl2 at pH 7.4. Brains were quickly removed and glued to the slicing stage of a vibrating microtome (Leica Microsystems), and 300 μm coronal slices containing the mPFC were cut between 3 and 4 mm anterior to bregma. Slices were incubated for 1 h at 37°C in oxygenated aCSF containing 126 mm NaCl, 10 mm glucose, 26 mm NaHCO3, 2.5 mm KCl, 1.25 mm NaH2PO4, 1 mm MgSO4, and 2 mm CaCl2 at pH 7.4. After incubation, the slices were allowed to equilibrate at room temperature for at least 1 h before recording.
6
Multimodal Neuroimmune Assessment
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At pre-determined time points post-injury (1, 3, or 7 DPI), brain and blood were collected. Approximately 300 μL of blood was collected by submandibular bleeds in BD Microtainer® MAP microtubes coated in ethylenediaminetetraacetic acid (Becton, Dickinson, and Company, Franklin Lakes, NJ). Blood (100 μL) was immediately processed for flow cytometry. The remaining blood (∼200 μL) was centrifuged to collect plasma for each mouse. Plasma samples were stored at −80°C until cytokine quantification. Immediately after blood collection, mice were injected (intraperitoneally) with Euthasol (0.002 mL/g; Patterson Veterinary, Greeley, CO) and perfused with ice-cold 1 × phosphate-buffered saline. Brains were removed and hemisected. One entire hemisphere was immediately processed for flow cytometry.
7
Tissue Collection and Processing for IHC and Histology
8
c-Fos Expression in Punished Rats
9
Murine Neuroimmune Dynamics Post-Injury
10
Ovariectomy Impact on Ewe Hormonal Regulation
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Thirty days after OVX, animals were placed into one of two groups, fed to maintain (FM) pre‐study body weight (n = 7) or feed‐restricted (FR) to lose 20% of pre‐study body weight by week 13 (n = 8), with animals from single, twin, and triplet pregnancies present in each group. All animals were weighed weekly, and the amount of feed was adjusted on an individual animal basis to achieve the desired body weight. Biweekly, blood samples (3–4 mL per sample) were taken via jugular venipuncture every 12 min for 4.5 h (276 min), and plasma samples were stored at −20°C until assayed for LH by a previously established radioimmunoassay.49 At the end of the experiment (week 13), ewes were given heparin (20,000 U, i.v.) and killed with an i.v. overdose of sodium pentobarbital (Euthasol; Patterson Veterinary). Heads were removed and perfused via the carotid arteries with four liters of 4% paraformaldehyde (PFA) in 0.1 m phosphate buffer (pH 7.4) containing 0.1% sodium nitrite. Blocks of hypothalamic tissue were removed and stored in 4% PFA for 24 h at 4°C and transferred to a 20% sucrose solution until sectioning. Frozen coronal sections were cut at 45–50 μm with a freezing microtome into a series of five vials and stored in cryopreservative solution until RNAscope and immunohistochemistry.
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