FISH 预处理

简短版方案不应替代试剂盒说明书。

预期用途

制备固定在载玻片上的细胞以用于使用 Vysis 探针的荧光原位杂交 (FISH)。

提供的试剂

试剂 数量 成分 储存
胃蛋白酶缓冲液 3 x 50 mL 10 mM HCl 2 至 25°C
蛋白酶 3 x 25 毫克 2500 - 3000 U/毫克蛋白酶,冻干粉 -20 至 8°C
PBS 2 x 250 mL  1X PBS 2 至 25°C
100X MgCl2 3 x 0.5 mL 2M MgCl2 * 6 H2O 2 至 25°C
20X SSC 1 x 66 克 氯化钠和柠檬酸钠 -25 至 30°C

提供的试剂
需要但不提供的材料

  • 无水乙醇 (EtOH)
  • 10% 缓冲福尔马林溶液
  • Carnoy 固定剂(甲醇:冰醋酸 (3:1))
  • 纯化水(蒸馏水或去离子水)
  • Coplin 染色缸
  • 15 mL 锥形离心管
  • 聚丙烯微量离心管 (1.5 mL)
  • 37°C 水浴

 

预处理程序

  1. 使玻片在室温下完全干燥。
  2. 在 73±1°C 温度下,将玻片浸入 2X SSC 中 2 分钟
  3. 将玻片浸入 37°C 的蛋白酶溶液中 10 分钟。(在加入 25 毫克(一管)蛋白酶之前,请确保缓冲液的温度为 37°C。)
  4. 在室温下用 1X PBS 洗涤玻片 5 分钟。
  5. 在室温下将玻片置于 1% 甲醛中固定 5 分钟。(将 12.5 mL 10% 中性缓冲福尔马林、37 mL 1X PBS 和 0.5 mL 100X MgCl2(一管)混合在一起。)
  6. 在室温下用 1X PBS 洗涤玻片 5 分钟。
  7. 在室温下将玻片浸入 70% 乙醇溶液进行脱水。使玻片在乙醇溶液中静置 1 分钟。使用 85% 乙醇重复上一步骤,然后再用 100% 乙醇重复上一步骤。
  8. 随后按照相应的 Vysis 探针方案继续进行。

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