Rats were anesthetized with isoflurane (R510-22, RWD, Shenzhen, Guangdong, China) inhalation (3–4% isoflurane for 3 minutes and maintenance with 1.5–2.5% of isoflurane until the end of operation). A 1.5-cm longitudinal incision was made on the rat’s skin along the posterior midline centered with the T10 centrum. To expose the spinal cord, we removed the T10 spinous process and lamina. A moderate SCI model was developed using an NYU Impactor Model III (W. M. Keck, Rutgers, NJ, USA) at a height of 25 mm and a weight of 10 g. Tail wagging and hind limb convulsion in rats indicated successful modeling. Muscle, subcutaneous tissue, and skin were sutured (Zhao et al., 2022). In the sham group, laminectomy was performed without SCI. Rats were held on a warm pad throughout the operation to maintain the body temperature until anesthesia recovery. After surgery, the bladder was expressed twice a day until recovery of urinary function.
R510 22
Manufactured by RWD Life Science
Sourced in China
The R510-22 is a laboratory equipment designed for scientific applications. It functions as a precision scale capable of measuring weights with high accuracy. The core function of this product is to provide reliable and precise weight measurements for research and experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
Laser doppler perfusion monitor, by Moor Instruments (2 mentions)
Rm2016, by Leica (1 mentions)
Pannoramic 250, by 3DHISTECH (1 mentions)
R540ip, by RWD Life Science (1 mentions)
Sprague dawley, by Charles River Laboratories (1 mentions)
Guiding cannula, by RWD Life Science (1 mentions)
Automatic microinjection system, by World Precision Instruments (1 mentions)
Sprague dawley rat, by Charles River Laboratories (1 mentions)
Legato 130, by RWD Life Science (1 mentions)
Nano zs90, by Malvern Panalytical (1 mentions)
Gentamycin sulfate, by Solarbio (1 mentions)
Cold light source, by World Precision Instruments (1 mentions)
Rose bengal, by Merck Group (1 mentions)
E8875, by Merck Group (1 mentions)
Super glue, by 3M (1 mentions)
Freezing microtome, by Leica (1 mentions)
Pannoramic scan, by 3DHISTECH (1 mentions)
Mp150 amplifier, by Biopac (1 mentions)
Sonos 7500, by Philips (1 mentions)
30 protocols using r510 22
1
Rat Spinal Cord Injury Model
2
Histological Analysis of Brain Tissue Damage
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Mice were anesthetized by 1% isoflurane (R510-22, RWD, China) using a gas anesthesia machine (R540IP, RWD, China) and subsequently perfused by injecting 4% paraformaldehyde (60 ml) through the heart. The brain was removed and fixed with 4% paraformaldehyde. The left damaged brain tissue was dissected to embed in paraffin and then sectioned using a microtome (RM2016, Leica, Germany) with a thickness of 5 μm. The sections were baked in an oven at 60 °C and dewaxed, and then stained with Harris hematoxylin for 3–8 min. After this, the sections were stained with eosin for 1–3 min after washing twice with tap water. Finally, the sections were dehydrated twice with 95% alcohol (5 min each) and washed three times with xylene (5 min each), and then fixed with natural gum. All sections were scanned with a digital pathology scanner system (Pannoramic 250, 3D HISTECH Ltd., Hungary), and 5 fields in injured areas were randomly selected for analysis. The output digital pathological images were 400x enlarged.
3
Measuring Colonic Mucus Thickness
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According to previous research [18 (link)], we made a horizontal perfusion chamber. For the ex vivo experiments, mice were anesthetized with isoflurane (R510-22, RWD Life Science Co., Ltd) and killed by cervical dislocation. The distal colon was dissected and flushed, and the muscle layer was removed. The mucosal tissue explant was mounted in a horizontal perfusion chamber. The upper surface of the colonic mucus was visualized by the addition of charcoal particles. The mucus thickness was determined by measuring the distance between the epithelial surface and the mucus surface by a micropipette attached to a patch clamp viewed through a stereomicroscope. Specifically, the height of the micropipette attached to the patch clamp was noted on the stereoscopic microscope using the homemade spatial positioning device. When the front end of the micropipette is located on the mucus surface, the corresponding height is recorded as H1. While keeping the horizontal position unchanged, when the front end of the descending micropipette falls into contact with the epithelial surface, the corresponding height is recorded as H2. H1 minus H2 to get the mucus thickness of the corresponding position. The mean value was calculated after repeated measurement for 3 times, then the position of the micropipette was changed and the mucus thickness at 6 different positions was measured.
4
Pancreas Tissue Investigation Protocol
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All mice were acclimatized for 30 minutes in a thermostatic laboratory at 26.0°C (standard deviation [SD], 0.5). After anesthetizing with 2% inhaled isoflurane (R510-22; RWD Life Science Co, Ltd, Shenzhen, China) in a 50% mixture of oxygen using small animal anesthesia machine (Matrx VMR; Midmark Corporation, Dayton, Ohio), mice were fixed on the operating plank in supine position with continued inhalation of volatile gas isoflurane. A designed 10-mm incision along the midline of the upper abdomen was made to expose the pancreas tissue and a stereotaxic holder was used to position and advance the probes within 1 mm upon the exposed pancreas tissue steadily. The temperature of pancreas tissue was monitored continuously to make sure it remained constant in the duration of the experiments.
5
SARS-CoV-2 Intranasal Infection in Mice
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All animals (mice) were inoculated intranasally with 50LD50 of the SARS-CoV-2/BMA8 strain after anesthetizing mice with isoflurane (R510-22, RWD, China). The body weight and living status of the mice were recorded every day. For MHC blocking experiments, H-2Kb mAbs (AF6-88.5.5.3, BioXCell, West Lebanon, NH) were used in C57BL/6N mice. The C57BL/6N mice were divided into 3 groups (n = 18 in each group): a control group (Control), a virus group (Virus), and an H-2Kb mAbs injection group (Virus + H-2Kb). Two days before the virus infection, H-2Kb mAbs (2 mg/mL/day) was injected intraperitoneally for 5 consecutive days in the H-2Kb mAbs injection group.
6
Auditory Function in Hypoxia-Exposed Rats
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The 15-day-pregnant Sprague-Dawley (SD) rats (n = 4) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Two hours (h) after the rats gave birth, all newborn rats were transferred together, with ten pups randomly divided into the control group (n = 5) and the IH group (n = 5), and the remaining pups applied for follow-up in vitro studies (n = 18). With reference to Alex’s treatment method [17 (link)], pups in the IH group were subjected to hypoxia treatment from 0 to 14 days after birth in a specially designed oxygen chamber. In brief, the pups were first exposed to 50% O2 for 30 min (min), followed by three hypoxia–reoxygenation treatments: the O2 concentration was reduced to 12% (lasted 1 min) and restored to 50% (lasted 10 min). The pups in the IH group received the above treatment once a day. For the rest of the time, the pups in the IH group and the control group were kept in a regular indoor air environment. Auditory brainstem response (ABR) thresholds were determined just before euthanization [18 ]. On the 56th day of birth, the SD rats in both groups were anesthetized with 4% isoflurane gas (R510-22; RWD, China). The intact cochlear tissue of the rats was taken out.
7
Stereotaxic Viral Vector Injection and In Vivo Peptide Administration in Mice
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For stereotaxic surgery, the 2-month-old C57BL/6 mice were placed in a stereotaxic apparatus and anesthetized with 2% isoflurane (RWD Life Science, R510-22) through a nose cone (300–500 mL/min, RWD Life Science, China, R500). Then, AAV-CaMKII-eGFP-2A-vector-3xFlag or AAV-CaMKII-eGFP-2A-GSK-3β WT-3xFlag or AAV-CaMKII-eGFP-2A-GSK-3β K15Q-3xFlag (1 μl, 4.0 × 1012 viral particles per ml) was bilaterally injected into the hippocampal CA1 region (posterior 1.82 mm, lateral 1.0 mm, and ventral 1.25 mm relative to bregma) at a rate of 0.1 μL/min. The needle was kept in place for 10 min before withdrawal. After the skin was sutured, the mice were placed on a heater for analepsia.
For in vivo peptide administration, guiding cannulas (RWD, Shenzhen, China) were implanted into the lateral ventricle (posterior 0.22 mm, lateral ± 1.0mm from the bregma, ventral -2.5 from the skull) of 12-month-old S129 mice and 3xTg mice, the peptides (1mM, 5 μL) were delivered using an automatic microinjection system (World Precision Instruments, USA), once every 2 days. Mice were restricted in a custom-designed device and stayed awake during drug administration. Upon deep anaesthesia (loss of the pedal pain, slowing of breathing and heart rate), these mice were euthanized by excising the heart for further analysis after behavioral experiments.
For in vivo peptide administration, guiding cannulas (RWD, Shenzhen, China) were implanted into the lateral ventricle (posterior 0.22 mm, lateral ± 1.0mm from the bregma, ventral -2.5 from the skull) of 12-month-old S129 mice and 3xTg mice, the peptides (1mM, 5 μL) were delivered using an automatic microinjection system (World Precision Instruments, USA), once every 2 days. Mice were restricted in a custom-designed device and stayed awake during drug administration. Upon deep anaesthesia (loss of the pedal pain, slowing of breathing and heart rate), these mice were euthanized by excising the heart for further analysis after behavioral experiments.
8
Ropivacaine Enhances Wound Healing in Rats
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In a total of 10 Sprague–Dawley rats (male, 200–250 g, 4-week-old) were purchased from Vital River (Beijing, China). All rats were housed at 25 °C with 12 h light and dark photocycle, food and water supplied. They were randomly divided into two groups (n = 5 per group), control and ropivacaine groups. All rats were first anesthetized with 2% ~ 3% isoflurane (R510-22, RWD Life Science, China) inhalation for induction and 1.5% isoflurane for maintenance. The back fur of the rats was carefully shaved for the area assigned for wounding. Excisional wounds with the size of 1 cm × 1 cm were created on each rat at day 0. A total of 1 ml containing 0.75% ropivacaine (ropivacaine group) or PBS (control group) was injected into the surrounding tissue of the wound once a day for 10 days. The wound areas were measured every 2 days. The rate of wound closure was calculated as follows: wound closure (%) = [(Day 0 wound area − Day X wound area)/Day 0 wound area] × 100%. All experimental procedures were approved by the Animal Ethics Committee of the Tianjin Nankai Hospital (Approval No. IRM-DWLL-2019042) and were performed in accordance with the National Institutes of Health “Guidelines for the Care and Use of Laboratory Animals”.
9
Stereotaxic Virus Injection and Optogenetic Manipulation
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The mice were anesthetized with isoflurane (5% for induction, 1–2.5% for maintenance, #R510-22, RWD, Shenzhen, China) and restricted using stereotaxic equipment (#68045, RWD). Erythromycin ointment was applied to the eyes of the mice to prevent dryness. We then made an incision on the mouse scalp to expose the skull before injecting the virus into the target brain area (LH: AP − 1.10 mm, ML − 1.05 mm, DV − 5.2 mm, mPFC: AP − 0.33 mm, ML + 2.10, DV − 2.10 mm, ACC: AP − 0.30 mm, ML + 1.25 mm, DV − 1.40 mm) using a glass pipette connected to a nanoliter injector with a controller (LEGATO 130, RWD) mounted on stereotaxic equipment. The volume of the injected virus was between 0.15 μl and 0.25 μl per side, and the speed was maintained at a rate of 0.05 μl/min. The pipette was removed 10 min after infusion. To enable optogenetic activation and in vivo calcium signal recording, an optic fiber (200 μm in diameter, 0.37 NA, Inper Tech, Hangzhou, China) with a ceramic ferrule was implanted into the mPFC or LH at the previously determined coordinates. To microinjection of drugs, a cannula (#62004, RWD) was implanted into the mPFC. The optic fiber and cannula were firmly secured to the skull using three skull screws (1 mm diameter, 3 mm length, Inper Tech) positioned around the craniotomy site, and dental cement was applied for additional stability.
10
Sirt1 Knockdown in Mouse Hippocampus
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The mice were randomly divided into three groups, each consisting of 14–16 mice: the untreated mice (control), the mice injected with AAV-CMV-EGFP (sham), and the mice injected with AAV-CMV-shSirt1-EGFP (shSirt1). The mice were anesthetized with inhalation of 2% isoflurane throughout the process by a small animal anesthesia machine (R510-22, RWD Life Science Co., Ltd., China) and fixed on a stereotactic apparatus (G1124701, RWD Life Science Co., Ltd., China). Both AAV-CMV-EGFP and AAV-shSirt1-EGFP were diluted to 3.5 × 1012 vp/mL. Bilateral injection with 1 µL of above AAV was performed into the dorsal hippocampal CA1 region, and stereotaxic coordinates were shown as follows: AP–2.00 mm, ± ML 1.5 mm, DV -1.0 mm from bregma. The injection rate was controlled at 100 nL/min. The needle syringe was left in place for about 10 min before being withdrawn. The scalp was sutured, disinfected with iodophor, and the mice were kept warm. After awakening from anesthesia, they were put back into the cage. After three weeks, the construction of Sirt1 knockdown in mouse hippocampus was considered successful [56 (link)].
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