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饿的神啊新虫 (著名写手)
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[求助]
急求！H2DCFHDA法测定叶片中H2O2含量 已有1人参与
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这个真的好纠结，试过好多方法，都不是很准确，打算用DAB染色或者H2DCFHDA荧光法做定性分析，用 Amplex red hydrogen peroxide/peroxidase assay kit (Molecular Probes, Eugene, OR)试剂盒做定量分析。需要同时做定量和定性吗，还是只做一个就可以了呢？ 我们实验室没有共聚焦显微镜，只有一个Varioskan Flash （Thermo），我百度之后发现它也是一个荧光酶标仪，可是我不知道该怎么处理植物叶片啊？百度过好多H2DCFHDA的试剂盒，比如那个活性氧检测试剂盒说是收集细胞后加载探针，可我这个是叶片啊，有没有战友做过类似的，给个指导吧！ |
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Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation Alexandre Grondin, Olivier Rodrigues, Lionel Verdoucq, Sylvain Merlot, Nathalie Leonhardt，and Christophe Maurela www.plantcell.org/cgi/doi/10.1105/tpc.15.00421 |
4楼2015-07-15 16:03:10
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感谢参与，应助指数 +1
饿的神啊: 金币+20, ★★★★★最佳答案 2015-07-15 16:38:10
感谢参与，应助指数 +1
饿的神啊: 金币+20, ★★★★★最佳答案 2015-07-15 16:38:10
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刚好在一篇PC上看到 Measurements of ROS Content Epidermal peels isolated using similar plants and procedures as for aperture measurements were incubated for 120 min under light (250 mEm22 s21) in a bathing solution as above (30 mM KCl, 1 mM CaCl2, and 10 mM MES/Tris, pH6.0). After 120 min, stomata were fully opened and 50 mM H2DCFDA (Sigma-Aldrich) was added to the bathing solution. This nonpolar, unreactive molecule diffuses intracellularly where it is deacetylated by endogenous esterase, to release H2DCF (Coelho et al., 2002; Foreman et al., 2003). H2DCF is then oxidized by H2O2, OH$ and possibly other ROS to highly fluorescent DCF (Schopfer et al., 2001, and references therein). After 20 min in the presence of H2DCFDA, epidermal peels were thoroughly washed four consecutive times with fresh bathing solution to eliminate all remaining extracellular H2DCFDA. To monitor ROS content of guard cells, a randomly chosen epidermal peal area containing 10 to 20 stained stomata was observed under constant light (250 mEm22 s21) using an epifluorescence microscope (Olympus IX70) equipped with a CCD camera (Andor Ixon +) connected to a computer operating Metafluor (Universal Imaging Corporation) image acquisition software. At t = 0, the bathing solution was equilibrated with 50 mM ABA or an equivalent concentration (0.5%) of ethanol (mock) and the fluorescence intensity was captured, in complete darkness, every 2 min for 30 min. Between each imaging process, the selected leaf area was reilluminated using the light source of the microscope. Average inner stomatal fluorescence was measured using ImageJ (http://rsb.info.nih.gov/ij/). www.plantcell.org/cgi/doi/10.1105/tpc.15.00421 |
2楼2015-07-15 01:25:27
饿的神啊
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3楼2015-07-15 09:40:43
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5楼2015-07-15 16:03:42
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