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Es, National Cancer Institute Peromyscus Genetic Stock Center, Columbia, SCReagents:Actin
Es, National Cancer Institute Peromyscus Genetic Stock Center, Columbia, SCReagents:Actin antibody Alcohol analytical kit Alda-1 ALT analytical kit Aminobenzotriazole BODIPY493/503 Bromosulfophthalein Calcein-AM Chemiluminescence kit Disulfiram ICN, Costa Mesa, CA BioVision, San Details science and library science), and psychology (psychiatry, neurosciences, and neurology Francisco, CA Dr. Paraffin sections right after paraformaldehyde fixation were stained with periodic acid Schiff (PAS) to assess glycogen.ImmunoblottingAcetaldehyde is extremely reactive, volatile and therefore tough to measure accurately. Nevertheless, acetaldehyde rapidly reacts with other aldehydes/proteins to type adducts. Such adducts are potentially toxic to mitochondria and linked to ethanol hepatotoxicity [23]. Accordingly, malondialdehyde-acetaldehyde adducts (MAA), a hybrid acetaldehyde adduct [23?5], and 4-hydroxynonenal (4-HNE) addu.Es, National Cancer Institute Peromyscus Genetic Stock Center, Columbia, SCReagents:Actin antibody Alcohol analytical kit Alda-1 ALT analytical kit Aminobenzotriazole BODIPY493/503 Bromosulfophthalein Calcein-AM Chemiluminescence kit Disulfiram ICN, Costa Mesa, CA BioVision, San Francisco, CA Dr. Daria Mochly-Rosen, Stanford University Pointe Scientific, Uncoln Park, MI Sigma-Aldrich, St. Louis, MO Invitrogen, Carlsbad, CA Sigma-Aldrich, St. Louis, MO Biotium Inc., Hayward, CA Pierce Biotec., Rockford, IL Sigma-Aldrich, St. Louis, MOEnliten ATP Assay Method Promega Corp., Madison, WI MAA antibody 4-HNE antibody Dr. Todd Wyatt, Univ. of Nebraska Healthcare Center Alpha Diagnostics, Inc., San Antonio, TXPolyethoxylated castor oil Sigma-Aldrich, St. Louis, MO Propidium iodide Rhodamine 123 Sigma-Aldrich, St. Louis, MO Sigma-Aldrich, St. Louis, MO Invitrogen, Carlsbad, CA Enzymatic Standbio, Boerne, TXMaterials and Techniques Animals and chemicalsSources for animals and reagents are listed in Table 1.TMRM Triglyceride analytical kitAnimals and ethanol treatmentMale C57BL/6 mice (eight? weeks), Cyp2E1 ull mice, and alcohol dehydrogenase (ADH)-positive and ADH-negative deer mice had access to chow diet plan ad libitum ahead of a single gavage with alcohol (1? g/kg) or automobile (saline). Some wild-type mice had been pretreated with a cytochrome P450 inhibitor, aminobenzotriazole (ABT, one hundred mg/kg, i.g.), an aldehyde dehydrogenase (ALDH) inhibitor, disulfiram (DSF, 200 mg/kg, i.p.), or an ALDH activator, Alda-1 [18] (N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide, 50 mg/kg, i.p.) 30 min before ethanol or with cyclosporin A (CsA, 10 mg/kg, i.g.) at 1 h just before ethanol. Cars for ABT, DSF, Alda-1 and CsA had been saline, DMSO, DMSO and 8.3 polyethoxylated castor oil with 8.three ethanol, respectively.Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; ALT, alanine aminotransferase; calcein-AM, calcein acetoxymethyl ester, 4-HNE, 4hydroxynonenal adducts; MAA, malondialdehyde-acetaldehyde adducts; TMRM, tetramethylrhodamine methylester. doi:ten.1371/journal.pone.0091308.tchloroform. Triglyceride was measured using an analytical kit (Table 1) [20].Hepatic ATP measurementTo assess the effects of ethanol on hepatic ATP, mice have been treated with saline or ethanol (6 g/kg, i.g.), and livers have been harvested 6 h later by freeze-clamping making use of aluminum tongs chilled in liquid nitrogen followed by storage at 280uC. ATP in trichloroacetic acid extracts was detected by luciferin-luciferase assay working with an Enliten ATP Assay Method (Table 1) [21,22].Clinical chemistry and histologyBlood was collected 1? h right after ethanol treatment. Alcohol and alanine aminotransferase (ALT) have been measured employing commercial analytical kits (Table 1) in line with the manufacturers‘ protocols.
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