9. Experimental procedures
For each experimental group, including controls, describe the procedures in enough detail to allow others to replicate them, including:
Essential information to describe in the manuscript includes the procedures used to develop the model (e.g. induction of the pathology), the procedures used to measure the outcomes, and pre- and post-experimental procedures, including animal handling, welfare monitoring and euthanasia. Animal handling can be a source of stress and the specific method used (e.g. mice picked up by tail or in cupped hands) can affect research outcomes [1-3]. Details about animal care and monitoring intrinsic to the procedure are discussed in further detail in item 16 – Animal care and monitoring. Provide enough detail to enable others to replicate the methods and highlight any quality assurance and quality control used [4,5]. A schematic of the experimental procedures with a timeline can give a clear overview of how the study was conducted. Information relevant to distinct types of interventions and resources are described below.
Pharmacological procedures (intervention and control)
Surgical procedures (including sham surgery)
Pathogen infection (intervention and control)
Reagents (e.g. antibodies, chemicals)
Equipment and software
Where available, cite the Research Resource Identifier (RRID) for reagents and tools used [7,8]. RRIDs are unique and stable, allowing unambiguous identification of reagents or tools used in a study, aiding other researchers to replicate the methods.
Detailed step-by-step procedures can also be saved and shared online, for example using Protocols.io , which assigns a Digital Object Identifier (DOI) to the protocol and allows cross-referencing between protocols and publications.
- Clarkson JM, Dwyer DM, Flecknell PA, Leach MC and Rowe C (2018). Handling method alters the hedonic value of reward in laboratory mice. Scientific reports. doi: 10.1038/s41598-018-20716-3
- Gouveia K and Hurst JL (2017). Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling. Scientific reports. doi: 10.1038/srep44999
- Hurst JL and West RS (2010). Taming anxiety in laboratory mice. Nat Methods. doi: 10.1038/nmeth.1500
- Hewitt JA, Brown LL, Murphy SJ, Grieder F and Silberberg SD (2017). Accelerating Biomedical Discoveries through Rigor and Transparency. ILAR journal. doi: 10.1093/ilar/ilx011
- Almeida JL, Cole KD and Plant AL (2016). Standards for Cell Line Authentication and Beyond. PLoS Biol. doi: 10.1371/journal.pbio.1002476
- Leary SL, Underwood W, Anthony R, Cartner S, Corey D, Grandin T, Greenacre C, Gwaltney-Bran S, McCrackin M, Meyer R, Miller D, Shearer J, Yanong R, Golab G and Patterson-Kane E (2013). AVMA guidelines for the euthanasia of animals: 2013 edition. https://www.avma.org/KB/Policies/Pages/Euthanasia-Guidelines.aspx
- Bandrowski AE and Martone ME (2016). RRIDs: A Simple Step toward Improving Reproducibility through Rigor and Transparency of Experimental Methods. Neuron. doi: 10.1016/j.neuron.2016.04.030
- Bandrowski A, Brush M, Grethe JS, Haendel MA, Kennedy DN, Hill S, Hof PR, Martone ME, Pols M, Tan SC, Washington N, Zudilova-Seinstra E and Vasilevsky N (2016). The Resource Identification Initiative: A Cultural Shift in Publishing. J Comp Neurol. doi: 10.1002/cne.23913
- Teytelman L and Stoliartchouk A (2016). Protocols.io: Reducing the knowledge that perishes because we do not publish it. Information Services & Use. doi: 10.1371/journal.pbio.100253
“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).” 
“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).” 
“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).” 
- Reynolds PS, Fisher BJ, McCarter J, Sweeney C, Martin EJ, Middleton P, Ellenberg M, Fowler E, Brophy DF, Fowler AA, 3rd, Spiess BD and Natarajan R (2018). Interventional Vitamin C: A Strategy for Attenuation of Coagulopathy and Inflammation in a Swine Polytrauma Model. The journal of trauma and acute care surgery. doi: 10.1097/ta.0000000000001844
- Bauters D, Bedossa P, Lijnen HR and Hemmeryckx B (2018). Functional role of ADAMTS5 in adiposity and metabolic health. PLoS One. doi: 10.1371/journal.pone.0190595
- Lian X, Wu X, Li Z, Zhang Y, Song K, Cai G, Li Q, Lin S, Chen X and Bai XY (2019). The combination of metformin and 2-deoxyglucose significantly inhibits cyst formation in miniature pigs with polycystic kidney disease. Br. J. Pharmacol. doi: 10.1111/bph.14558
Clearly report the frequency and timing of experimental procedures and measurements, including the light and dark cycle (e.g. 12L:12D), circadian time cues (e.g. lights on at 08:00), and experimental time sequence (e.g. interval between baseline and comparator measurements or interval between procedures and measurements). Along with innate circadian rhythms, these can affect research outcomes such as behavioural, physiological, and immunological parameters [1,2]. Also report the timing and frequency of welfare assessments, taking into consideration the normal activity patterns (see item 15 – Animal care and monitoring). For example, nocturnal animals may not show behavioural signs of discomfort during the day .
If the timing of procedures or measurements varies between animals, this information can be provided as a supplementary table listing each animal.
- Bartlang MS, Neumann ID, Slattery DA, Uschold-Schmidt N, Kraus D, Helfrich-Förster C and Reber SO (2012). Time matters: pathological effects of repeated psychosocial stress during the active, but not inactive, phase of male mice. Journal of Endocrinology. doi: 10.1530/joe-12-0267
- Paul AK, Gueven N and Dietis N (2017). Morphine dosing strategy plays a key role in the generation and duration of the produced antinociceptive tolerance. Neuropharmacology. doi: 10.1016/j.neuropharm.2017.04.034
- Hawkins P, Morton DB, Burman O, Dennison N, Honess P, Jennings M, Lane S, Middleton V, Roughan JV, Wells S and Westwood K (2011). A guide to defining and implementing protocols for the welfare assessment of laboratory animals: eleventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Laboratory Animals. doi: 10.1258/la.2010.010031
“Blood pressure, heart rate, oxygen saturation and amount of blood extracted were recorded every 5 minutes. Blood samples were drawn at baseline (pre injury), 0 minutes (immediately after injury), and after 30 and 60 minutes.” 
“After a 5-h fast (7:30–12:30am), awake and freely moving mice were randomized and subjected to three consecutive clamps performed in the same mice as described above, with a 2 days recovery after each hyperinsulinemic/hypoglycemic (mHypo, n = 6) or hyperinsulinemic/euglycemic (mEugly, n = 4) clamps.” 
- Hagemo JS, Jørgensen JJ, Ostrowski SR, Holtan A, Gundersen Y, Johansson PI, Næss PA and Gaarder C (2013). Changes in fibrinogen availability and utilization in an animal model of traumatic coagulopathy. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine. doi: 10.1186/1757-7241-21-56
- Emery M, Nanchen N, Preitner F, Ibberson M and Roduit R (2016). Biological Characterization of Gene Response to Insulin-Induced Hypoglycemia in Mouse Retina. PLOS ONE. doi: 10.1371/journal.pone.0150266
Physiological acclimatisation after a stressful event, such as transport (e.g. between supplier, animal facility, operating theatre and laboratory), but before the experiment begins allows stabilisation of physiological responses of the animal [1,2]. Protocols vary depending on species, strain, and outcome; for example physiological acclimatisation following transportation of different animals can take anywhere from 24 hours to more than one week . Procedural acclimatisation, immediately before a procedure, allows stabilisation of the animals’ responses after unaccustomed handling, novel environments, and previous procedures, which otherwise can induce behavioural and physiological changes [4,5]. Standard acclimatisation periods may vary between research labs and this information cannot be inferred by readers.
Indicate where studies were performed (e.g. dedicated laboratory space or animal facility, home cage, open field arena, water maze) and whether periods of physiological or procedural acclimatisation were included in the study protocol, including type and duration. If the study involved multiple sites, explicitly state where each experiment and sample analysis was performed. Include any accreditation of laboratories if appropriate (e.g. if samples were sent to a commercial laboratory for analysis).
- Holmes AM, Emmans CJ, Coleman R, Smith TE and Hosie CA (2018). Effects of transportation, transport medium and re-housing on Xenopus laevis (Daudin). Gen Comp Endocrinol. doi: 10.1016/j.ygcen.2018.03.015
- Conour LA, Murray KA and Brown MJ (2006). Preparation of animals for research--issues to consider for rodents and rabbits. ILAR J. doi: 10.1093/ilar.47.4.283
- Obernier JA and Baldwin RL (2006). Establishing an appropriate period of acclimatization following transportation of laboratory animals. ILAR Journal. doi: 10.1093/ilar.47.4.364
- Krahn DD, Gosnell BA and Majchrzak MJ (1990). The anorectic effects of CRH and restraint stress decrease with repeated exposures. Biological psychiatry. doi: 10.1016/0006-3223(90)90046-5
- Pitman DL, Ottenweller JE and Natelson BH (1988). Plasma corticosterone levels during repeated presentation of two intensities of restraint stress: chronic stress and habituation. Physiology & behavior. doi: 10.1016/0031-9384(88)90097-2
“Fish were singly housed for 1 week before being habituated to the conditioning tank over 2 consecutive days. The conditioning tank consisted of an opaque tank measuring 20 cm (w) 15 cm (h) 30 cm (l) containing 2.5 l of aquarium water with distinct visual cues (spots or stripes) on walls at each end of the tank…During habituation, each individual fish was placed in the conditioning apparatus for 20 minutes with free access to both compartments and then returned to its home tank.” 
- Brock AJ, Goody SMG, Mead AN, Sudwarts A, Parker MO and Brennan CH (2017). Assessing the Value of the Zebrafish Conditioned Place Preference Model for Predicting Human Abuse Potential. The Journal of pharmacology and experimental therapeutics. doi: 10.1124/jpet.117.242628
There may be numerous approaches to investigate any given research problem, therefore it is important to explain why a particular procedure or technique was chosen. This is especially relevant when procedures are novel or specific to a research laboratory, or constrained by the animal model or experimental equipment (e.g. route of administration determined by animal size ).
- Turner PV, Brabb T, Pekow C and Vasbinder MA (2011). Administration of substances to laboratory animals: routes of administration and factors to consider. Journal of the American Association for Laboratory Animal Science : JAALAS. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189662/
“Because of the very small caliber of the murine tail veins, partial paravenous injection is common if 18F-FDG is administered by tail vein injection (intravenous). This could have significantly biased our comparison of the biodistribution of 18F-FDG under various conditions. Therefore, we used intraperitoneal injection of 18F-FDG for our experiments evaluating the influence of animal handling on 18F-FDG biodistribution.” 
“Since Xenopus oocytes have a higher potential for homologous recombination than fertilized embryos…we next tested whether the host transfer method could be used for efficient HDR-mediated knock-in. We targeted the C-terminus of X. laevis Ctnnb1 (β-catenin), a key cytoskeletal protein and effector of the canonical Wnt pathway, because previous studies have shown that addition of epitope tags to the C-terminus do not affect the function of the resulting fusion protein (Fig…)…CRISPR components were injected into X. laevis oocytes followed by host transfer or into embryos.” 
- Fueger BJ, Czernin J, Hildebrandt I, Tran C, Halpern BS, Stout D, Phelps ME and Weber WA (2006). Impact of animal handling on the results of 18F-FDG PET studies in mice. Journal of nuclear medicine : official publication, Society of Nuclear Medicine. https://jnm.snmjournals.org/content/47/6/999.long
- Aslan Y, Tadjuidje E, Zorn AM and Cha SW (2017). High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus. Development. doi: 10.1242/dev.152967