采用 PAP 染色的 FISH:溶液制备

采用 PAP 染色的 FISH:溶液制备
  • 制作胃蛋白酶储备溶液。保持冷冻。将 0.5 g 胃蛋白酶溶于 5 mL 蒸馏水中。胃蛋白酶浓度为 100 毫克/ml。
  • 配制胃蛋白酶工作液 (0.05 毫克/ml)。在 40 mL 的 10mM 盐酸中加入 20 ul 胃蛋白酶储备溶液。胃蛋白酶最终浓度为 0.05 毫克/ml。
  • 1% 甲醛固定溶液 (V=400 ml)。将 10 mL 37% 甲醛溶液与 386 mL PBSx1 和 4 mL 100XMgCl2 混合,其中 100xMgCl2 = 2 M MgCl2。储存于冰箱中。每次使用后弃置。每次使用后弃置溶液。
 
 
 
 
取下盖玻片
  1. 将玻片在室温下的二甲苯中浸泡约 48 小时,或直到盖玻片易于从载玻片上取下或脱落。
  2. 将玻片在新鲜二甲苯中洗涤 5 分钟。建议在新鲜溶液中进行第三次二甲苯洗涤,洗涤 5 分钟。
  3. 风干玻片约 15 分钟
  4. 将玻片放入含有 95% 乙醇的 Coplin 染色缸中。在冰箱中可以储存数天/数星期。
  5. 将玻片放入含有 85% 乙醇的 Coplin 染色缸中洗涤,然后在含有 70% 乙醇的 Coplin 染色缸中洗涤,每次洗涤 5 分钟。
  6. 将玻片放入 2xSSC 中孵育 15-60 分钟。随后进行预处理,然后进行 FISH。
 
 
 
 
预处理程序
  1. 将玻片放入 37ºC 下的 2 X SSC 中孵育 10 分钟。
  2. 将玻片放入 37ºC 下的胃蛋白酶工作液中孵育 13 分钟。
  3. 将玻片放入室温下的 PBS X1 中孵育 5 分钟。
  4. 将玻片在室温下的 1% 甲醛中固定 5 分钟。
  5. 将玻片放入室温下的 PBS X1 中洗涤 5 分钟。
  6. 根据 FISH 方案,继续进行玻片变性。
 
 
 
 

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