石蜡预处理 3

 

简短版方案不应替代试剂盒说明书。

预期用途
制备固定在带正电荷载玻片上的石蜡包埋组织切片,以用于采用 Vysis 多色 DNA FISH 探针的荧光原位杂交 (FISH*)。

摘要和原则
以下程序旨在使用 Vysis 多色 DNA FISH 探针(例如 ProVysionTM 多色探针,订购编号 32-131090)时能够最大限度地提高 FISH 对组织的处理能力和渗透性。此预处理测定将有助于最大限度地减少 DNA 探针杂交后的重颗粒背景,该背景在特定组织类型中可能有时会出现。
paraffin-pretreatment-3-table.gif

样品玻片制备
备注:使用福尔马林固定了 24-48 小时的样本。

  1. 用切片机切取 4-5 µm 厚的石蜡切片。
  2.  将切片漂浮于 40ºC 的纯化水(即三蒸水)的水浴中。
  3. 在带正电荷的载玻片上固定切片。
  4. 风干玻片,并在 56ºC 温度下烘烤过夜。

将玻片脱蜡

  1. 将玻片浸入室温下的 Hemo-De 中 5 分钟。
  2. 重复第一步两次,每次使用新的 Hemo-De。
  3. 将玻片置于室温下的 100% 无水乙醇中脱水 1 分钟。重复。
  4. 如果需要,将玻片风干 2-5 分钟。

玻片/蛋白酶预处理

  1. 将玻片浸入室温的 45% 甲酸/0.3% 过氧化氢中 15 分钟。
  2. 将玻片浸入纯化水中 3 分钟。用纸巾吸干玻片边缘,以去除多余水分。
  3. 将玻片浸入 80ºC 的预处理溶液中 10 分钟。
  4. 在新鲜的纯化水中洗涤 3 分钟。
  5. 在 37°C 蛋白酶工作溶液中孵育 10 分钟。(精确地)量取 50 mL 蛋白酶缓冲液 III 并置于 37±1°C 的水浴中。在使用前请确保缓冲液的温度为 37±1°C。通过向 515 µL 纯化水中加入 9.0 毫克(一管)蛋白酶来制备蛋白酶储备溶液。

    要制备蛋白酶工作溶液
    ,将 100 µL 蛋白酶储备溶液移入(预热且精确测量体积的)一瓶 50 mL 蛋白酶缓冲液 III 中。冷冻剩余的蛋白酶储备溶液。
  6. 在蛋白酶停止溶液中孵育 3 分钟以停止反应。
  7. 在纯化水中洗涤玻片 3 分钟。
  8. 将玻片风干 2-5 分钟。
  9. 随后按照相应的 Vysis 探针方案继续进行。

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