Prior to brain dissection, adult mice were anesthetized by intraperitoneal injections with about 1 ml of 2.5% 2,2,2-tribromethanol (Sigma-Aldrich) and intracardially perfused with 4% paraformaldehyde (PFA). The whole brain was therefore extracted and the fixation continued in 4% PFA overnight at 4 °C. After cryoprotection in 30% sucrose, brains were cryo-sectioned at 40 μm of thickness, and slices encompassing the SVZ and the olfactory bulbs (OB) were analyzed by immunohistochemistry.
2 2 2 tribromethanol
Manufactured by Merck Group
Sourced in United States
2,2,2-tribromethanol is a chemical compound with the molecular formula C(CH3)3Br3. It is a colorless crystalline solid that is used as a laboratory reagent and intermediate in various organic synthesis reactions.
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7 protocols using 2 2 2 tribromethanol
1
Brain Fixation and Cryosectioning
2
Brain Sample Preparation for Western Blot
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Animals were sacrificed immediately after finishing the experiment. All mice were deeply anesthetized by intraperitoneal injection of 2,2,2-tribromethanol (Sigma-Aldrich, St. Louis, USA) followed by transcardial perfusion with cold 0.1% phosphate-buffered saline (PBS). Brain samples were isolated and frozen in liquid nitrogen. For the Western blot analyses, the brain samples from four randomly selected animals from each subgroup were chosen, giving a total of 32 animals. On the day of testing, tissues were homogenized in RIPA buffer containing protease inhibitors (Roche Applied Science, Penzberg, Germany). SDS-containing reducing sample buffer was added giving a final concentration of 1x [43 (link)]. Probes were denatured for 5 min at 60°C.
3
Folic Acid-Induced Kidney Injury in Transgenic Mice
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The adult TG lines A and D and wild-type (WT) male littermates aged 4 to 5 months were used. TG and WT mice were injected i.p. with 250 mg/kg body weight of FA (Sigma F7876), dissolved in the vehicle 0.3 M sodium bicarbonate (veh). Control animals, both WT and TG, received 0.3 ml of veh. Additional studies were done in transgenic homozygous mice from line A, injected with FA or vehicle (used as control, because there were not wild type littermates). Spot urine and serum were collected on days 0, 7 and 14 from all of the animals and analyzed for proteinuria and creatininuria using Bradford assay (Bio-Rad) and a Creatinina Wiener Lab Kit (Wiener Laboratorios, Rosario, Argentina), respectively. Seven or 14 days after the injection, the animals were anesthetized with 2% 2,2,2-tribromethanol (Sigma) dissolved in 2-methyl-buthanol (Sigma). The kidneys were removed, decapsulated and cut along the sagittal plane. The left kidney was fixed in 4% formaldehyde, while the right kidney was immediately frozen in liquid nitrogen and processed for RNA and protein extraction. The specimens were embedded in paraffin and cut into 4 µm tissue sections for further histological (PAS/Masson) and IHC studies using antibodies against gremlin and αSMA, F4/80, CD3 and PCNA.
4
Continuous NBM Dosing via Osmotic Pumps
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Dosing to the NBM was achieved using Alzet osmotic minipumps (Charles River), which allowed the continuous administration of hNGFp over a period of 28 days. The daily dose was equivalent to the intranasal dose: 0.54 µg/kg. Control animals were represented by 5xFAD mice implanted with a mini-osmotic pump releasing PBS. The mini-osmotic pumps were prepared according to the manufacturer’s instructions. On the day of surgery, mice were anaesthetized with 10 μl/g of body weight of a 2.5% solution of 2,2,2-tribromethanol (Sigma-Aldrich). Minipumps were implanted into the brain in proximity of the NBM according to the following coordinates: bregma: −0.6 mm, lateral to sagittal suture: 2.1 mm, depth: 3.5 mm.
5
Intestinal Contractility Modulation by CFTR Activators
6
Intranasal BDNF administration in AD11 mice
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BDNF (Harlan) administration was performed on anaesthetized mice as described in De Rosa et al. [20 (link)]. Briefly, 2,2,2-tribromethanol (Sigma–Aldrich, St. Louis, Mo) was dissolved in absolute ethanol at the concentration of 1 g/ml and stored at 4 °C in the dark. After dilution in 0.9% NaCl at the final concentration of 1.5%, it was injected i.p. at the dosage of 250 mg/kg of body weight to induce anaesthesia, which followed within 5–10 min after injection. After anaesthesia, mice were laid on their back, with the head in upright position, as described before [19 , 20 (link), 22 (link), 28 (link)]. A solution of BDNF in 0.1 M phosphate-buffered saline (PBS, pH 7.4) was administered intranasally to AD11 mice at the dose of 12.6, 42 and 420 pmol (0.3, 1 and 10 µM), The solution was delivered 3 μl at a time, alternating the nostrils, with a lapse of 2 min between each administration, for a total of 14 times. During these procedures, mice were kept on their back and the nostrils were always kept open. As control, AD11 mice were treated with PBS. Administrations were repeated for 7 times, at alternate days, for a total of 15 days.
7
Bilateral Ovariectomy in Rodents
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Animals were anesthetized by intraperitoneal administration of 2.5% solution of 2,2,2-tribromethanol (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 400 mg/kg and bilateral ovariectomy was performed under aseptic conditions using microsurgery technique. The animals of the same age which had only incisions of the abdominal cavity and isolation of the ovaries without resection (sham-operated) were used as a control group. Wounds were sutured in layers; the animals were kept under a heat lamp until the recovery from anesthesia.
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