The Vanderbilt University Medical Center Institutional Animal Use and Care Committee approved all experimental procedures. Young adult (2 mo) C57 mice (C57BL/6, male; Charles River Laboratory) were maintained in a 12-h light/dark cycle with standard rodent chow and water available ad libitum. We used unilateral microbead occlusion to elevate IOP in C57 mice, with the fellow eye receiving an equal volume saline injection as internal control (16 (link)–18 (link, link)). We measured IOP biweekly using rebound tonometry (Tono-Pen XL; Medtronic Solan), as described (16 (link)–18 (link, link)) (Fig. S9 ). For physiological recordings and intracellular filling, retinas were dissected under long-wavelength light (630 nm, 800 µW/cm2; Ushio FND/FG). A subset of animals was bilaterally injected intravitreally with 1 μL of 0.5 mg cholera toxin subunit B (CTB) conjugated to Alexa Fluor 488 (Invitrogen). Intact transport within serial coronal superior colliculus sections was quantified (18 (link), 23 (link)). For tissue required for immunolabeling of vertical sections and analysis of anterograde transport, mice were perfused transcardially with 4% paraformaldehyde.
C57bl6 male
Manufactured by Charles River Laboratories
Sourced in United States
C57bl6 male is a commercially available strain of laboratory mouse. This strain is commonly used in biomedical research due to its well-characterized genetic and physiological characteristics.
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11 protocols using c57bl6 male
1
Microbead Occlusion Model for Elevated IOP in Mice
2
Social Defeat Stress in Mice
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C57bl6 male (n = 50) and female (n = 28) mice aged 7–8 weeks old and CD1 males aged 4–6 months old were obtained from Charles River Laboratories (USA). In total 50 males and 28 females were subjected to social defeat. Mice were housed under a 12 h light/dark cycle at 22–24 °C with no water and food restriction. CD1 mice were individually housed except during social defeats. All other mice were group-housed (4/cage) before and singly housed after social defeats. Laval’s University Institutional Animal Care Committee, in respect with the Canadian Council on Animal Care guidelines, approved all experimental procedures. The study was carried out in compliance with the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357 ).
3
Evaluation of AT-RvD1 in Bacterial Pneumonia
4
Evaluation of AT-RvD1 in Bacterial Pneumonia
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Live gram-negative bacteria (E. coli or P. aeruginosa, 1 × 106 CFU in 25 μl saline) were instilled selectively into the left mainstem bronchus of anesthetized mice (C57bl/6, male, 8–10 weeks; Charles River) as in16 (link). Mice received AT-RvD1 (100 ng, IV) or vehicle (<0.1% (vol/vol) ethanol in saline) 1 hr after intra-bronchial E. coli or P. aeruginosa. In select experiments, mice received ciprofloxacin (0.2, 2, or 20 mg/kg, IP) or vehicle in addition to AT-RvD1, 1 hr after intra-bronchial E. coli. In separate experiments, animals were treated with AT-RvD1 (100 ng, IB) or vehicle via a repeat tracheostomy surgery 24 and 36 hrs after high-dose intra-bronchial E. coli (2×106 CFU). A high dose of E. coli was used to induce neutrophil infiltration comparable to LPS-induced lung inflammation. In pathogen-mediated acute lung inflammation experiments, animals were treated with E. coli LPS (0.3 mg/kg, 50 μl in saline, IB). In sterile acute lung inflammation experiments, hydrochloric acid (HCl, 0.1N, pH ~1.0, 50 μl IB) was administered as in17 (link).
5
Experimental Mice Study Protocol
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Experiments with mice (C57BL/6 male, 6–12 weeks old; Charles River, Wilmington, MA, USA) were carried out according to a protocol approved by the Institutional Animal Care and Use Committee of Georgia College & State University (Approved 11 April 2019). Animals were housed (5 animals maximum per cage) and food and water were available ad libitum. Animal husbandry was done according to the Guide for the Care and Use of Laboratory Animals (8th edition; National Academies Press, Washington, DC, USA).
6
Murine C57/Bl6 Animal Husbandry
7
Circadian Rhythms in Mouse Liver
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All animal experiments and procedures were approved by the Swiss Veterinary Office (authorisation VD-1453.4). C57BL/6 male were purchased from Charles River. Bmal1−/− mice were a kind gift from Dr. Frédéric Gachon and were generated as previously described [63] (link), [64] (link). 12–14 week old (at time of sacrifice), mice were housed in a 12 h light/12 h dark (LD) regimen for 2 weeks with food and water freely available during night and day. They were then phase-entrained to a 12 hr/12 hr LD regimen with food access between ZT12 and ZT24 for 7 days (ZT = Zeitgeber time; ZT0 is defined as the time when the lights are turned on and ZT12 as the time when lights are turned off). At each ZT2, ZT06, ZT10, ZT14, ZT18 and ZT22 three to five mice were anesthetized with isoflurane and decapitated. Mice were killed under dim red light at ZTs during the dark phase. The livers were perfused with 2 ml of PBS through the spleen and immediately collected. A small piece of liver tissue was snap-frozen in liquid nitrogen. The remaining liver tissue was immediately homogenized in PBS containing 1% formaldehyde for chromatin preparation.
8
Fecal Microbiota Transplantation: Histidine Effects
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We leveraged data from our previous fecal microbiota transplantation experiment.1 (link) Briefly, fecal samples from low- (n = 2) and high- (n = 2) histidine donors matched for age and BMI were suspended in sterile reduced PBS (N2 gas and thioglycolic acid, Sigma Aldrich, St. Louis, MO). Eight mice (8-week-old C57BL/6 male, Charles River) per patient were treated with an antibiotic mixture for 7 days (neomycin, ampicillin, metronidazole) and after a 4-day washout, mice were administered 20 mg/day of fecal matter for 4 consecutive days. Two weeks later, mice were sacrificed, and liver and plasma were collected and frozen. Experimental groups were randomly allocated.
9
Chronic Social Defeat Stress in Mice
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C57bl6 male (n = 49) and female (n = 43) mice aged 7-8 weeks old and CD1 males aged 4-6 months old were obtained from Charles River Laboratories (USA). Mice were fed ad libitum at 22-25°C on a 12-hour light/dark cycle. CD-1 mice were singly housed except during social defeats. All other mice were housed in groups (4/cage) before and singly housed after social defeat episodes. In total, 35 males and 30 females were subjected to CSDS. Fourteen males and 13 females served as controls. All experimental procedures were approved by Université Laval's Institutional Animal Care Committee in respect with the Canadian Council on Animal Care guidelines.
10
Social Defeat Stress in Mice
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C57bl6 male (n = 50) and female (n = 28) mice aged 7-8 weeks old and CD1 males aged 4-6 months old were obtained from Charles River Laboratories (USA). In total 50 males and 28 females were subjected to social defeat. Mice were housed under a 12 hours light/dark cycle at 22-24ºC with no water and food restriction. CD1 mice were individually housed except during social defeats. All other mice were grouphoused (4/cage) before and singly housed after social defeats. All experimental procedures were approved by Laval's University Institutional Animal Care Committee in respect with the Canadian Council on Animal Care guidelines.
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