FISH 用于石蜡包埋的分离核

  • 此技术适用于已固定很长时间的样品或难以用作 FISH 靶标的样品(如脑样品)。
  • 使用二甲苯(或 Histoclear)将 40 µm 组织切片脱蜡 3 次,每次 10 分钟。
  • 在 100% 无水乙醇中脱水 2 次,每次 5 分钟。
  • 在 37°C 的 4 毫克/ml 胃蛋白酶(Sigma P-7012,在 0.9% NaCl 中,pH 1.5)中孵育 2 小时。每 30 分钟涡旋一次。
  • 通过 40 µm 尼龙网过滤。
  • 以 800 rpm (标准台式仪器)旋转 5-10 分钟。
  • 用 PBS 洗涤沉淀,以 800 rpm 旋转 2 次,每次 5-10 分钟。
  • 用尽可能最少量的 PBS 重悬沉淀。
  • 涡旋细胞核,涂在 FISHer Superfrost Plus(类别编号12-550-15)载玻片上,使其在 65 °C 下干燥 10 分钟。
  • 在 PBS 中洗涤载玻片。
  • 在含 0.01% Triton 100 的 PBS 中孵育 1.5 分钟。
  • 在 PBS 中洗涤 3 次,每次 3 分钟。
  • 在含 0.3 毫克/ml 链霉蛋白酶的 50 mM Tris/Cl (pH 7.6)、5 mM EDTA 中孵育 5 分钟。
  • 在含 2 毫克/ml 甘氨酸的 PBS 中孵育 3 次,每次 3 分钟。
  • 在含 4% 多聚甲醛的 PBS 中孵育 5 分钟。
  • 在含 2 毫克/ml 甘氨酸的 PBS 中孵育 3 分钟。
  • 在 30%、60%、80%、95% 和 100% 无水乙醇中各洗涤 3 分钟。
  • 风干玻片。
  • 在 90°C 的烘箱中使探针和靶标 DNA 同时变性 1-3 分钟。
  • 在 37°C 的加湿盒中孵育至少 1 小时至过夜。
  • 在 45°C 的以下洗涤溶液中各洗涤 1 分钟:3 x 50% 甲酰胺/2XSSC,在 2X SSC、2X SSC/0.1% NP-40、2X SSC/0.1% NP-40(室温)中各洗涤一次。
  • 本实验方案需要甲酰胺和二甲苯,它们是致畸剂。请参阅相应的 MSDS(物料安全数据表)以了解更多信息。

 

实验方案提交人:

Dr. Rizwan Naaem - 细胞遗传学实验室主任

Baystate 医疗中心,马萨诸塞州斯普林菲尔德,1994

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