2 methylbutane

All animal experiments were performed in accordance with the institutional guidelines and approved by the Internal Committee for the Care and Use of Laboratory Animals of the Center for Research and Advanced Studies of the National Polytechnic Institute under number 0001–02 and following the guidelines of the Official Mexican Standard NOM-062-ZOO-1999. Male Fischer-344 rats weighting 180 to 200 g, from the Production Unit of Experimental Laboratory Animals (UPEAL-CINVESTAV, Mexico), were feed ad libitum and housed in a controlled environment (12 h light/12 h dark cycle; at 22 ± 2°C, and humidity at 55 ± 10%). For carcinogenesis, a modified resistant hepatocyte model was used [10 (link)]. Rats were initiated with diethylnitrosamine (DEN) (200 mg/kg of body weight) at day 0. Then, 2-acetylaminofluorene (AAF) was administered (20 mg/kg per dose) at days 7, 8 and 9, followed by 3/5 partial hepatectomy (PH) at day 10 (Fig 1). Three groups of non-treated animals were sacrificed by exsanguination on the first day and at 9 and 18 months after the beginning of the experiment. Treated animals were sacrificed at 1, 7, 11 and 16 days and at 1, 9 and 18 months. Their livers were excised, washed in physiological saline solution, frozen with liquid nitrogen in 2-methyl butane (Sigma-Aldrich) and stored at -80°C.

Three weeks after transplantation, all rats were deeply anaesthetized with an overdose of sodium pentobarbital (intraperitoneally) and transcardially perfused with 0.1 mol L⁻¹ cold PBS, followed by 4% paraformaldehyde (diluted in 0.1 mol L⁻¹ PBS at pH 7.4) to fix the brains. The brains were then removed, suspended in 4% paraformaldehyde for 24 hour at 4°C and then transferred to the graded sucrose solutions (10%, 20% and 30%), dissolved in 0.1 mol L⁻¹ PBS, and then frozen using 2‐methylbutane and stored in the −80°C freezer until they were sectioned coronally (30 µm) on a cryostat (Vibratome UltraPro 5000) set at −20°C. Brains from rats used for Western blot analysis were directly removed without perfusion and the fresh tissue was flash‐frozen using 2‐methylbutane (Sigma) and stored at −80°C until further use.

Skin samples from normal fetuses collected at the slaughterhouse, and from affected (haired and hairless areas) and unaffected cows were embedded in Optimal Cutting Temperature compound (OCT) and were snap frozen by immersion in 2-methylbutane (59080; Sigma—Aldrich), which was chilled in liquid nitrogen. The frozen tissue blocks were stored at -80°C until cutting. Immunohistochemistry was carried out as previously described [50 ]. Briefly, cryostat sections were fixed in ice-cold acetone for 3 min and endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in methanol for 30 min. A protein block was obtained by applying 10% normal goat serum in PBS for 30 min. The slides were incubated with the primary antibody (the in-house produced polyclonal rabbit antibody) at 4◦C overnight. The positive reactions were detected with a LSAB-kit (Dako) according to the manufacturer’s instructions.

Quadriceps muscles were embedded in Optimal Cutting Temperature compound (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen-cooled 2-methylbutane (Sigma, St. Louis, MO, USA), and stored at -80° C. Transverse 7 μm-thick frozen sections were cut with a Leica cryostat and mounted onto SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA). Muscle sections were dried for 2 hours at -20° C, and then stored at -80° C until analysis. Frozen quadriceps sections were removed from -80° C and fixed in 4% paraformaldehyde (PFA) for 15 minutes. After being permeabilized and blocked, the sections were incubated with rabbit anti-laminin antibody (L9393, Sigma-Aldrich) overnight at 4° C, followed by goat-anti-rabbit cross-adsorbed secondary antibody Alexa Fluor 488 (A11008, Invitrogen by Thermo Fisher Scientific) for 1 hour in a humidified chamber at room temperature. Sections were mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen by Thermo Fisher Scientific). Images were captured at 10x magnification with a Zeiss Axio Imager microscope. Myofiber cross-sectional area, demarcated by Laminin staining, was quantified using MuscleJ [68 (link)].

SOD1^G93A mice and wild-type littermates were sacrificed by inhalation of CO₂ for muscle analysis. The extraocular muscles and lumbrical muscles (from the plantar surface of the hind-paw) were dissected in 0.1 M PBS and fixed in 4% PFA (Sigma-Aldrich) for 10 min for NMJ analysis or snap frozen in 2-Methylbutane (Sigma-Aldrich) on dry ice for immunoblotting. Only muscles innervated by CNIII were included in the extraocular analysis (superior rectus, inferior rectus, medial rectus, and inferior oblique). Staining for NMJ analysis was done as previously described6 (link) using antibodies detailed in Table 1. For CNS immunohistochemistry, animals were anesthetized with avertin (2,2,2-Tribromoethanol; Sigma-Aldrich) and perfused intracardially with PBS followed by 4% PFA. Brains and spinal cords were dissected and postfixed (for 3 hours and 1 hour, respectively), cryoprotected in sucrose and sectioned (30 μm). All CNS tissues were stained as previously described12 (link) using the following primary antibodies (Table 1) and counter stained with NeuroTrace 435/455 Blue Fluorescent Nissl Stain (1:200 in PBS; Life Technologies) for 30 min. Tissues were imaged on a Zeiss LSM700 confocal microscope.

Analytical grade acetonitrile, methanol and formic acid were obtained from Fisher Scientific (Loughborough, Leicestershire, UK). 2-methylbutane was obtained from Sigma-Aldrich (Poole, Dorset, UK). Test compounds were obtained in house from AstraZeneca compound management group (Macclesfield, Cheshire, UK) with the exception of moxifloxacin and SCH-23390 which were purchased from Sigma-Aldrich (Poole, Dorset, UK) and clozapine-d4 which was purchased from Qmx Laboratories (Thaxted, Essex, UK).