9. Experimental procedures What was done, how it was done, and what was used. examples

[1]

“Fig … shows the timeline for instrumentation, stabilization, shock/injury, and resuscitation…Animals were food-deprived for 18 hours before surgery, but allowed free access to water. On the morning of surgery, swine were sedated with tiletamine-zolazepam (Telazol®; 5-8 mg/kg IM; Zoetis Inc., Kalamazoo MI) in the holding pen, weighed, and masked with isoflurane (3%, balance 100% O2) for transport to the lab. The marginal ear vein was catheterized for administration of atropine (0.02 mg/kg IV; Sparhawk Laboratories, Lenexa KS), and buprenorphine for pre-emptive analgesia (3 mg/ml IV; ZooPharm, Laramie WY). Ophthalmic ointment (Puralube®; Fera Pharmaceuticals) was applied to prevent corneal drying. Animals were intubated in dorsal recumbency with a cuffed 6 or 7 Fr endotracheal tube. Anesthetic plane was maintained by isoflurane (1-1.5%; 21-23 % O2, balance N2). Oxygen saturation (sPO2, %) and heart rate (HR) were monitored with a veterinary pulse oximeter placed in the buccal cavity (Masimo SET Radical-7; Irvine CA). Core temperature was monitored with a rectal probe and maintained at 36.5-38oC with a microprocessor-controlled feedback water blanket (Blanketrol® II, Cincinnati Sub-Zero (CSZ) Cincinnati, OH) placed under the animal. Anesthetic depth was assessed every 5 min for the duration of the experiment by reflexes (corneal touch, pedal flexion, coronary band pinch) and vital signs (sPO2, HR, core temperature).” [1]

“For the diet-induced obesity (DIO) model, eight-week-old male mice had ad libitum access to drinking water and were kept on standard chow (SFD, 10.9 kJ/g) or on western high-fat diet (HFD; 22 kJ/g; kcal from 42% fat, 43% from carbohydrates and 15% from protein; E15721-34, Ssniff, Soest, Germany) for 15 weeks (https://dx.doi.org/10.17504/protocols.io.kbacsie).” [2]

“The frozen kidney tissues were lysed. The protein concentration was determined with the Pierce BCA assay kit (catalogue number 23225; Thermo Fisher Scientific, Rockford, IL, USA). A total of 100–150 μg total proteins were resolved on a 6–12% SDS-PAGE gel. The proteins were then transferred to a nitrocellulose membrane, blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against the following proteins: proliferating cell nuclear antigen (PCNA; Cat# 2586, RRID: AB_2160343), phospho-AMPK (Cat# 2531, RRID: AB_330330), phospho-mTOR (Cat# 2971, RRID: AB_330970)…The β-actin (Cat# A5441, RRID: AB_476744) antibody was obtained fromSigma. The blots were subsequently probed with HRP-conjugated anti-mouse (Cat# A0216) or anti-rabbit IgG (Cat# A0208; Beyotime Biotechnology, Beijing, China) at 1:1000. Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed using ImageJ software (RRID: SCR_003070, Bio-Rad Laboratories).” [3]