羊水细胞样本处理

  1. 将 2 - 5 mL 全羊水 (AF) 标本在 1,000 RPM 下离心 5 分钟。标本不应出现血迹或呈现褐色。 
  2. 将沉淀重悬于 2 - 5 mL 1X 胰蛋白酶/EDTA(0.05% 胰蛋白酶,0.53 mM EDTA 4Na 的 Hanks 平衡盐溶液,不含 CaCl2、MgCl2 6H2O 和 MgSO4 7H2O),并在 37ºC 水浴中孵育至少 15 分钟。
  3. 将悬浮物在 1,000 RPM 下离心 5 分钟。
  4. 将沉淀重悬于 2 - 5 mL 0.56% KCl 中,并在 37ºC 水浴中孵育 20 分钟。
  5. 向细胞/低渗溶液中加入 0.8 - 2 mL 固定剂(3:1 甲醇:冰醋酸),轻轻涡旋。
  6. 将悬浮物在 1,000 RPM 下离心 5 分钟,然后在 1 mL 新鲜固定剂中重悬沉淀。将固定的标本在 4ºC 下储存至少 30 分钟或直到准备进行 FISH。如需长期储存,将固定的标本储存在固定剂中,于 -20ºC 温度下保存。
  7. 将细胞置于载玻片前,根据细胞沉淀物的大小调节细胞悬液的体积。如果需要,特别是长时间储存(> 1 个月)后,在玻片制备前用固定剂洗涤沉淀。
  8. 为了制备 FISH 玻片,将细胞悬浮液直接滴在 1 或 2 个冷载玻片上,形成 2 个杂交区域(每个区域 15 - 25 µL 细胞悬浮液)。

 然后使用 FISH 预处理试剂盒

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