GUS Protocol (fool-proof version)
Solutions for GUS staining:
Staining Buffer (final concentrations): (make to u, do not prepare ahead of time)
0.2% Triton X-100
50mM NaHPO4 Buffer (pH7.2)
2mM Potassium Ferrocyanide
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2mM Potassium Ferricyanide
Water to volume
note: higher ferri and ferrocyanide concentrations give lower overall staining level, but more specificity. 2mM works well for most applications, but the concentrations may need to be adjusted for certain needs.
Stock solutions that can be made ahead of time:
10% Triton X-100
0.5M NaHPO4 出国留学读研流程Buffer (pH7.2)
100 mM Potassium Ferrocyanide (Store in the dark at 4°C)
100mM Potassium Ferricyanide (Store in the dark at 4°C) 2013英语四级改革
100mM X-Gluc (5-bromo-4-chloro-3-indolyl ß-D-glucuronide cyclohexamine salt) in DMF
FAA (can be made ahead and stored at room temperature)
50% Ethanol
5% Formaldehyde 全国翻译专业资格水平考试
10% acetic acid
water to volume
pray1. Harvest tissue and place in cold 90% Acetone on ice.This should stay on ice until all samples are harvested. For sample containers, eppendorf tubes and glass scintillation vials work well. 将样品收集于冰上预冷的90%丙酮中。不能用聚丙烯管装。
2. 英语学习在线发音When all samples are harvested, place at room temperature for 20 minutes. 室温放置20分钟。
3. 速度与激情7 票房Remove acetone from the samples, and add staining buffer on ice. 吸去丙酮，用染色buffer（不含X-Gluc）清洗
4. Add X- Gluc to the staining buffer to a final concentration of 2mM - from a 100mM stock solution of X-Gluc in DMF- this must be kept in the dark at -20°C . 配置染色液。X-Gluc终浓度为2mM.
5. Remove staining buffer from samples and add staining buffer with X-Gluc on ice. 吸去染色buf.，加入染色液
6. Infiltrate the samples under vacuum, on ice, for 15 to 20 minutes. Relea the vacuum
slowly and verify that all the samples sink. If they don't, infiltrate again until they all sink to the bottom when the vacuum is relead. 抽真空15-20 min.
7. Incubate at 37°C (I usually do it overnight, but it depends on transgene strength. It is not advisable from my experience to go too long (over two days) as the tissue ems to begin deteriorating during long incubations. 37丝光沸石度，过夜
8. Remove samples from incubator and remove staining buffer. Go through an Ethanol ries in which samples are incubated successively in 20%, 35% and 50% ethanol at room temperature for 30 minutes each. 乙醇脱色
9. Incubate in FAA (recipe below) for 30 minutes at room temperature to fix the tissue.
10. Remove FAA and add 70% ethanol. At this point the tissue can be stored at 4°C for long periods, or examined under the microscope
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