29 The compounds were dissolved in deionized water and administered in 5 ��L on PND 1, 10 ��L on PND 8 and 20 ��L on PND 15. The mice were weaned on PND 21, culled to 24�C26 male B6C3F1 mice per treatment group, and monitored for 1 year. Throughout the study, the mice (both the dams and the pups) were fed, ad libitum, irradiated Purina 5LG6 pellets, C646 order
a diet low in acrylamide (<50 ppb) compared to other commercial formulations.32 One mouse from the 0.70 mmol glycidamide per kg body weight treatment group was mis-sexed and discarded. Two additional mice from this group were inadvertently used for microbiological surveillance. These three mice were not examined further. The animals were monitored for 1 year after treatment. At the termination of the study, all surviving animals were euthanized by exposure to carbon dioxide, gross examinations were performed, and all gross lesions visible at necropsy were recorded, including number, location, size and color. Gross observations predominantly involved the liver, a primary target tissue in the neonatal mouse bioassay.33�C35 The livers and lungs were removed and dorsal and ventral views of these organs were recorded using digital imagery before they were preserved in 10% neutral buffered formalin. The livers were trimmed, processed and embedded in infiltrating media (Formula R?), sectioned at approximately 5 ��m, and stained with hematoxylin and eosin <a href="http://www.selleckchem.com/products/obeticholic-acid.html
">Obeticholic Acid in vivo for histopathological examination. Complete necropsies were also performed on animals that died naturally or that were submitted moribund before the scheduled terminal sacrifice. Genomic DNA was isolated from the formalin-fixed and paraffin-embedded normal and tumor tissue samples. Briefly, paraffin-embedded liver tissue sections were deparaffinized by incubation with xylene for 30 min at 45��C, further washed with 100% ethanol, and subsequently rehydrated in 90% and 70% ethanol. Tissue pellets were digested overnight with 40 ��g of proteinase K (New England Biolabs, Ipswich, MA) in 400 ��L of DNA extraction buffer Y 27632
(500 mM Tris�CHCl, 1 mM EDTA, 5 mM NaCl and pH 8.0). DNA was further recovered by phenol:chloroform:isoamyl alcohol extraction. H-ras codons 12, 13 and 61 mutations were analyzed using the method of Mitchell and Warshawsky.36 Briefly, primary polymerase chain reaction (PCR) amplification of DNA was performed and the PCR products were further digested with BstNI (codon 12), BglI (codon 13) and BclI (codon 61) restriction endonucleases (New England Biolabs). Digested PCR products were subjected to a second amplification, followed by a second digestion with the restriction endonucleases used previously. The digested products were then separated by electrophoresis on a 2.5% agarose gel. Mutant bands were excised from the gel, re-amplified using nested primers and sequenced (Retrogen, San Diego, CA).