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发布于:2018-9-14 12:05:12  访问:28 次 回复:0 篇
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N 2.five glutaraldehyde, 1 formaldehyde in 0.1 M sucrose, <a href="https://www.ncbi.nlm.nih.gov/pubmed/28461567" title=View Abstract(s)">PubMed ID:https://www.ncbi.nlm.nih.gov/p
Grids have been stained in 1 uranyl acetate, 50 methanol for 15 minutes, rinsed twice (30 CS-4708 site seconds every single) with 100 ethanol followed by 50 ethanol/water (15 seconds), 30 ethanol/water (15 seconds), and four 15 second washes in water. They then have been rinsed 3 times (10 minutes each) at 4 with 0.2 M Na-cacodylate, pH 7.four, after which post-fixed for 90 minutes, at four , with 0.five OsO4, 0.5 KFe(CN)6 and 0.1 M Na-cacodylate, pH PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29069523 7.4, for 90 minutes on ice. Subsequent methods had been carried out at space temperature. Worms then have been rinsed 3 occasions, ten minutes every single, in 0.1 M Na-cacodylate buffer, stained in 1 uranyl acetate in 0.1 M sodium acetate, pH 5.two, for 1 hour at room temperature, followed by three 5-minute 0.1 M sodium acetate washes and 3 5-minute distilled water washes. Worms have been packed in parallel in a Vshaped plexiglass trough and have been embedded 3 seaplaque agarose. Around 1 mm2 blocks then were dehydrated in acetone and embedded in Embed 812 [42]. For every genotype examined, no less than three person animals had been sectioned, and various sections from each and every animal along the entire gonad span have been analyzed. Crosssections of worms have been cut applying a diamond knife and Leica microtome and were picked up on carbon-overformvar coated single hole grids. Sections had been dried overnight then stained applying minor modifications of your Hall (1995) procedure. Stains and washes had been ready in 16 well plastic culture dishes at room temperature. Grids have been stained in 1 uranyl acetate, 50 methanol for 15 minutes, rinsed twice (30 seconds every single) with 100 ethanol followed by 50 ethanol/water (15 seconds), 30 ethanol/water (15 seconds), and four 15 second washes in water. Sections then were stained for five minutes with 0.1 lead citrate in 0.1 M NaOH, rinsed twice with 0.02 M NaOH (1 minute/change), rinsed 5 instances in water (15 seconds/wash) and have been air-dried ahead of examination using the TEM.DAPI and phalloidin staining of dissected gonads The gonads of 12-hour-old adult hermaphrodite animals had been dissected and fixed with three paraformaldehyde containing 0.1 M K2HPO4, pH7.2, for 1 hour at space temperature. The specimens had been washed after with phosphatebuffered saline (PBS) with 0.1 Tween-20 (PBT) for five minutes followed by remedy with one hundred methanol for 5 minute at -20 . Specimens had been treated with PBS containing 100 ng/ 4‘,6-diamidino-2-phenylindole (DAPI) or rhodamine-phalloidin for 10 minutes at area temperature followed by 3 washes in PBT. Gonads were mounted on a two agarose pad in M9 buffer with 1 mg/ml 1,4-diazabicyclo [2.two.2]octane (DABCO) antifade reagent. BrdU labeling of dissected gonads Escherichia coli strain MG1693 (a thymidine-deficient E. coli strain kindly offered by the E. coli stock center) were grown minimal medium (M9) with 0.four glucose, 1 mMPage 17 of(web page quantity not for citation purposes)BMC Developmental Biology 2008, eight:http://www.biomedcentral.com/1471-213X/8/MgSO4, 1.25 /ml vitamin B1, 0.5 thymidine, and 10 bromodeoxyuridine (BrdU) overnight at 37 [25].
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