All cells were grown in Gibco αMEM (Life Technologies, Grand Island, NY) with 10% fetal bovine serum (Biosera, Kansas City, MO) and antibiotics. Primary bone marrow was isolated according to standard protocols. In brief, mice were sedated with isofluorane (Baxter, Deerfield, IL) and sacrificed by cervical dislocation. Bone marrow cells were collected by washing the marrow cavity αMEM delivered via a 21 G needle. Cells were cultured overnight and the non-adherent fraction was separated and allowed to expand for 3 days in 30 ng/mL CSF-1 (R&D Systems, Minneapolis, MN). BMMs were harvested and plated with 30 ng/mL CSF-1 and 10 ng/mL RANKL (R&D Systems) for differentiation into osteoclasts.
Isofluorane
Manufactured by Baxter
Sourced in United States, Germany
Isoflurane is an inhalational anesthetic agent used in veterinary medicine. It is a colorless, nonflammable, and volatile liquid that is administered through inhalation. Isoflurane provides general anesthesia and is commonly used in surgical procedures to induce and maintain unconsciousness in animals.
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Lab products found in correlation
Vt1000s vibratome, by Leica (2 mentions)
Csf 1, by R&D Systems (1 mentions)
Rankl, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
80045rnae50, by Sino Biological (1 mentions)
β galactosidase, by Abcam (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Te 1000, by Nikon (1 mentions)
Fitc or cy3 labeled secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Coup tfii, by Abcam (1 mentions)
α sma, by Merck Group (1 mentions)
Propofol, by Fresenius (1 mentions)
Thinpro insulin syringe, by Terumo (1 mentions)
Egg yolk lecithin, by Merck Group (1 mentions)
Naringin, by Extrasynthese (1 mentions)
Neohesperidin, by Extrasynthese (1 mentions)
Oleuropein, by Extrasynthese (1 mentions)
Hydroxytyrosol, by Extrasynthese (1 mentions)
Rbc lysing buffer, by Merck Group (1 mentions)
Nylon cell strainer, by BD (1 mentions)
19 protocols using isofluorane
1
Osteoclast Differentiation from Murine Bone Marrow
2
Anesthesia and MRI Monitoring in Healthy Rats
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Seventy-eight healthy Han-Wistar rats (Crl:WI; males; 200–225 g) were obtained from Charles River (Sulzfeld, Germany). The animals were kept under standard laboratory conditions at a temperature of 22°C and a dark/light rhythm of 12 hours. Standard rat chow and water were provided ad libitum. The animals were handled and treated according to German animal regulations. The GBCA administration and the MRI were performed in anesthesia initiated with 4% isofluorane and maintained with 1.5% isofluorane (Baxter GmbH, Unterschleißheim, Germany). During the MRI, the body temperature was maintained with a circulating warm water pad.
3
BALB/c Mice Intranasal Influenza Infection
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Eight to ten week old female BALB/c mice were maintained in the Specific Pathogen Free Bioresources Facility at the Department of Microbiology and Immunology (University of Melbourne). Infectious agents were diluted in sterile PBS and 50 μL administered intranasally to anaesthetized mice (inhaled 2.5% isofluorane; Baxter Healthcare). Mice were monitored daily for weight loss and illness. The infectious dose for all experiments was 100 plaque-forming units (PFU). At 72 h post-infection (hpi), bronchioalveolar lavage (BAL) fluid and lung samples were extracted for analysis of cellular content and viral load. Viral RNA from these samples was sequenced to confirm no further mutations had occurred in vivo. Viral load was assessed via homogenization of whole lung tissue and determined by the quantitation of plaques on confluent MDCK cell monolayers as previously described [12 ].
4
In vivo Bioluminescence Imaging of Cerebral Malaria
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Visualization and quantification of luciferase activity in whole bodies or isolated brains of P. berghei ANKA infected and uninfected WT and ApoE−/− mice was achieved using an intensified-charge-coupled device (I-CCD) video camera and in vivo Imaging System (IVIS 100, Xenogen)60 (link). The mice were anesthetized by using isofluorane (Baxter), shaved and were injected intraperitoneally with 5 mg/ml D-Luciferin (Caliper Life Sciences). Measurements were performed within 3 to 5 min after the injection of D-Luciferin. Luciferase-expressing regions were visualized using the in vivo Imaging system. The mice were euthanized and the brains were placed in a Petri dish and imaged. The bioluminescence was quantified by selecting a region of interest and using average radiance (p/s/cm2/sr) as the unit.
5
Preparation of Acute Cortical Slices
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Animals were deeply anaesthetized using isofluorane (Baxter Corporation) and subsequently euthanized via decapitation. Brains of the animals were quickly extracted and submerged into a frozen choline dissection buffer. The buffer consisted of the following: 119.0 mM choline chloride, 2.5 mM KCl, 4.3 mM MgSO4, 1.0 mM CaCl2, 1.0 mM NaH2PO4, 1.3 mM sodium ascorbate, 11.0 mM glucose, 26.2 mM NaHCO3, and was perfused using carbogen (95% O2/5% CO2). Acute cortical slices containing the PFC were produced using a Leica VT1000S vibratome. The brain was sliced coronally at a thickness of 300 µm. Once the slices were collected, they were placed in a recovery chamber filled with a standard artificial cerebrospinal fluid (ACSF) consisting of 119.0 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 1.0 mM NaH2PO4, 11.0 mM glucose, and 26.2 mM NaHCO3. The ACSF was continuously perfused using carbogen (95% O2/5% CO2) and maintained at a temperature of 37 °C. Following slicing, the chamber was left to recover for 1 h prior to experiments where it equilibrated to room temperature.
6
Developmental Timing of Social Stress Exposure
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Social stress or control manipulation was begun during 1 of 3 developmental periods: early adolescence (PD 30–36), mid-adolescence (PD 42–46), or adulthood (PD 69–76). These adolescent ages were selected to span the social and physical stages of early and mid-adolescence as designated previously (Spear 2000 (link); McCormick and Mathews 2010 (link); Sturman and Moghaddam 2011 (link)). Social stress and control rats were exposed to 5 days of their respective experimental manipulation. Twenty-four hours after completion of the final manipulation, rats were sacrificed using inhaled 5% isofluorane (Baxter Healthcare) until they no longer responded to toe pinch, perfused intracardially with ice-cold sucrose-rich artificial cerebrospinal fluid (aCSF) in order to improve tissue slice quality, decapitated, and brains were removed and prepared for slice recordings.
7
PET Imaging of Zr-89 Labeled Antibodies
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PET imaging experiments were conducted on a microPET Focus 120 scanner (Concorde Microsystems). Mice were anesthetized by inhalation of 1.5–2% isofluorane (Baxter Healthcare) in an oxygen gas mixture 10 min before recording PET images. PET data for each group (n = 4) was recorded, with mice under isofluorane anesthesia (1.5–2%), in list mode at 4, 8, 24, and 48 h after intravenous injection of [89Zr]Zr-DFO-trastuzumab or [89Zr]Zr-DFO-IgG. Images were analyzed using ASIPro VM software (Concorde Microsystems).
8
Rat Intervertebral Disc Degeneration Model
9
Wound Healing in Mice Under Corticosteroid
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Excisional wounds were carried out in C57BL6 mice and/or LysCre‐MR KO mice with or without topical pretreatment once a day with 125 μg clobetasol (Clarelux, 500 μg·g−1, GlaxoSmithKline laboratory, Rueil‐Malmaison, France) on the back for 10 days before wounding. Mice were initially anaesthetized with 4.9% isofluorane (Baxter, Newbury, UK) at 300 ml/min ambient air flow, which was reduced to 2% for maintenance. After shaving, we selected the dorsal areas featured by typical pink telogen stage colour. Excisional 6‐mm diameter wounds were generated on the back of the mice using a disposable device (Kai Europe GmbH, Solingen, Germany). No dressings were applied on the wounds. Treatment consisted in twice daily applications on the wound surface of 30 μl of the MR antagonist potassium canrenoate [0.5 mM]) or PBS until killing (Day 5). Photographs of the mouse backs with wounds were done at various timepoints after wounding using a digital camera (Sony CybershotH 10.1‐megapixel DSC‐W180). The wound surface was calculated as the percentage of unhealed area/initial wound area at Days 3 and 5.
10
Histological Analysis of Murine Kidneys
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Mice were anesthetized with isofluorane (Baxter) and subsequently perfused via the left ventricle with 4°C PBS for 1 minute. Kidneys from adult mice were fixed with 4% paraformaldehyde (PFA), dehydrated and embedded in paraffin. Hematoxylin/eosin, PAS and Masson's trichrome staining were performed using standard protocols (Yang et al., 2010) .
Immunofluorescence staining of mouse kidneys was performed on paraffin sections as previously described (Yang et al., 2010) . Briefly, the tissue sections were deparaffinized, followed by antigen retrieval and rehydration. Then tissue antigens were labeled with primary antibodies to COUP-TFII (Abcam, diluted 1: 200), αSMA (Sigma, 1:400), collagen1 (EMD Millipore, 1:400), CD31 (Abcam 1: 200), PDGF receptor beta (PDGFRβ, Abcam 1:400), or βgalactosidase (Abcam 1:100), followed by FITC or Cy3-labeled secondary antibodies (Jackson ImmunoResearch). Some immunostaining was performed on frozen sections. Cryosections (7 µm) were fixed in 4% PFA for 2 hours, and then, washed in 30% sucrose solution overnight. Primary antibodies used in cryosections recognized the following proteins: CD3 (eBioscience 1:100), LyG6, F4/80 (ThermoFisher), Images were captured by a confocal (Nikon C1) or standard fluorescent microscope (Nikon TE 1000).
Immunofluorescence staining of mouse kidneys was performed on paraffin sections as previously described (Yang et al., 2010) . Briefly, the tissue sections were deparaffinized, followed by antigen retrieval and rehydration. Then tissue antigens were labeled with primary antibodies to COUP-TFII (Abcam, diluted 1: 200), αSMA (Sigma, 1:400), collagen1 (EMD Millipore, 1:400), CD31 (Abcam 1: 200), PDGF receptor beta (PDGFRβ, Abcam 1:400), or βgalactosidase (Abcam 1:100), followed by FITC or Cy3-labeled secondary antibodies (Jackson ImmunoResearch). Some immunostaining was performed on frozen sections. Cryosections (7 µm) were fixed in 4% PFA for 2 hours, and then, washed in 30% sucrose solution overnight. Primary antibodies used in cryosections recognized the following proteins: CD3 (eBioscience 1:100), LyG6, F4/80 (ThermoFisher), Images were captured by a confocal (Nikon C1) or standard fluorescent microscope (Nikon TE 1000).
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