Ut below a protocol authorized by the Institutional Animal Care and Use Committee at Baylor College of Medicine and were in accordance using the National Institutes of Health suggestions for the care plus the use of laboratory animals. Mice had been maintained inside a temperaturecontrolled facility with 12 h light/dark cycles and absolutely free access to standard chow and water. Male mice, 8-12 weeks old, were utilized for this study. To induce fatty liver, mice had been injected with tunicamycin (0.5 mg/kg body weight). Just after tunicamycin injection, liver tissues had been collected at 0, 24, and 48 h, flash-frozen in embedding medium containing a 3:1 mixture of Tissue Freezing Medium (Triangle Biomedical Sciences) and gum tragacanth (Sigma-Aldrich) at -80 , and sectioned to 10 m-thick using Cryostat (Leica CM 3000). C. elegans Strains and Culture. Wild-type N2 strain from the Caenorhabditis Genetics Center had been grown on standard NGM plates (containing 5 mg/L cholesterol) with E. coli OP50 at 20 employing typical protocols.22 Deuterated Fatty Acid Supplementation. McA-RH7777 liver cells were supplemented with 0.4 mM palmitic acid-D31 (PA-D31) or oleic acid-D34 (OA-D34) for 7 h. The fatty acid/BSA answer was ready as described ahead of, after which supplemented to culture medium. For C. elegans fatty acid supplementation, OP50 bacterial culture was mixed well with four mM PA-D31, or OA-D34, or two mM PA-D31 + 2 mM arachidonic acid (AA-D8), and then seeded onto NGM plates. Following 1-day-old adult worms were fed for the desired time, they had been mounted onto two agarose pads with 0.five NaN3 as anesthetic on glass microscope slides. Thin Layer Chromatography (TLC). For yeast TLC, ten OD units of yeast cells had been broken with glass beads and lipids have been extracted with chloroform:methanol (1:1 v/v). Lipids were dried below nitrogen gas, loaded onto TLC plates (Whatman), created in hexane:ethyl ether:acetic acid (80:20:1 v/v), and charred with cupric sulfate until lipid bands had been visible. For C. elegans TLC, about 5000 worms for each sample had been collected and homogenized. Total lipids have been extracted and loaded onto silica gel G TLC plates (Analtech), created in hexane:diethyl ethyl:acetic acid (75:25:two v/v). The TLC plate was stained with 0.05 Primuline (Sigma-Aldrich, in acetone:H2O, eight:2 v/v).Perfluoropropionic anhydride Chemscene Lipids were visualized employing a Gel Imaging Technique (Bio-Rad) with fluorescence (Ex 488 nm/Em 555 nm).tert-Butyl 4-bromopicolinate Purity For mouse liver TLC, 200 mg liver tissues for every single sample had been homogenized in PBS and utilized for lipid extraction.PMID:33586576 TLC plates have been created in petroleum ether:ether:glacial acetic acid (85:25:1 v/v) and visualized in saturated iodine chamber.Final results Quantitatively Differentiating Two Classes of Neutral Lipid Applying hsSRS Imaging. To test the capability of hsSRS in imaging unique lipid molecules, we very first made use of this technique to acquire the spectra of cholesteryl oleate, triolein, cholesterol and oleic acid in deuterated chloroform solutions. We acquired a total of 20 photos that were evenly spaced in between 2825 and 3050 cm-1 for every single sample, which covers the C-H stretching region of most biological molecules and supplies spectra at a wavenumber spacing of 12.five cm-1 (Figure 1b). The four lipid molecules lack characteristic chemical bonds needed to separatedx.doi.org/10.1021/ja504199s | J. Am. Chem. Soc. 2014, 136, 8820-Journal of the American Chemical Society them when employing traditional SRS, but they produce distinguishable spectra when imaged with hsSRS (Figure 1b). Interestingly, we found that.