Sigma Trap染色说明书.docVIP

1、Prewarm sufficient deionized water for a day’s use to 37℃, check temperature before use. 2、Bring Fixative solution to room temperature (18-26). Fix slides by immersing in Fixative Solution for 30 seconds. Rinse thoroughly in deionized water: don’t allow slides to dry. 3、To each of 2 test tubes add 0.5 ml Fast Garnet GBC Base solution and add 0.5 ml Sodium Nitrite Solution. Mix by gentle inversion for 30 seconds. Let stand 2 minutes. 4、Label two 100-ml beakers A and B and add the following while mixing. ? Beaker A Beaker B Deionized water prewarmed to 37℃ 45ml 45ml Diazotized Fast Garnet GBC solution from step 3 1.0ml 1.0ml Naphthol AS-BI Phosphate Solution 0.5ml 0.5ml Acetate Solution 2.0ml 2.0ml Tartrate Solution ------ 1.0ml 5、Label Coplin jars A and B and transfer solutions from beakers to appropriate Coplin jar. Warm solutions in jars to 37℃ in water bath. Check that temperature is at 37℃ before adding slides. 6、Add slides to Coplin jars and incubate 1 hour in 37℃ water bath protected from light. 7、After 1 hour, rinse slides thoroughly in deionized water, then counterstain 2 minutes in Hematoxylin Solution, Gill No.3. 8、Rinse several minutes in alkaline tap water to blue nuclei. 9、Air dry and evaluate microscopically. Coverslipping is not recommended since dye fades with time. TRAP染色（sigma-aldrich.NO387） 1. 37℃预热足够的去离子水。 2. Fixation solution（固定液）室温放置，细胞爬片浸没30秒，用去离子水彻底清洗，不要让爬片太干燥。（或细胞爬片后，用多聚甲醛（最好37°效果好些）固定20分钟）。吸弃时注意易将细胞吸掉 2.1 个人意见：1xPBS（37°) 洗一次，吸弃时注意易将细胞吸掉,建议用200μ枪， 3. 准备两个1.5 mL ep管，各加5?L Fast garnet GBC Base solution（快速石榴石型碱溶液）和5?L Sodium Nitrite Solution（亚硝酸盐溶液） ,轻轻混匀30秒，室温静置2分钟。两者混合后为步骤三混匀的溶液 4. 另标签一个管为A（B），并按以下要求配置混合液：(个人意见：说明书的 B 不用做只是对照) A 步骤三混匀的溶液 10 ?L Naphthol AS-BI Phosphate Solution 5?L （萘酚AS-BI磷酸溶液） Acetate Solution 20?L （醋酸盐溶液） 37℃预热的去离子水 450?L Tartrate Solution 10?L （酒石酸盐溶液） （个人意见：最后加酒石酸盐溶液 避光） 5. 把步骤4中的配置的染色液倒入合适的染色缸中（个人意见：直接在35mm皿上放置可拆卸96微孔板染，然后避光（锡箔纸包裹后放置于一次性手套）水浴），水浴加热到37℃，再加玻片前一定要保证温度达到37℃。 6. 把爬片放入染色缸中，37℃避光孵育1小时。 7. 孵育1