人极体细胞的制备

本程序在去除极体后实施。通过机械分离,从未受精和受精的卵母细胞中获取极体细胞进行活检。

程序

  1. 配制固定剂(甲醇:冰醋酸为 3:1),并储存在冰箱中,直至使用。
  2. 准备装有甲醇的 Coplin 染色缸。
  3. 在 35 x 10 mm 皮氏培养皿底部雕刻一个小圆圈。圆圈标识放置极体的目标区域,以更易于重新定位。
  4. 将标本标识和相关信息写在显微镜载玻片的磨砂区域上。用固定剂和无绒布擦拭载玻片进行预清洁。
  5. 用纯化水填充悬滴载玻片。
  6. 使用切片机加热 25 µL 毛细移液管的尖端,同时拉动加热的移液管尖端,使其伸出并形成内径为 50 µm 的尖端。确保移液管的延伸尖端有一个均匀的裂口,这样就不会锋利。锐利尖端可能损伤极体。如有必要,再次折断尖端以创制平滑尖端。移液管开口的尺寸应在拾取极体之前检查,以确保其尺寸适当;如果直径过大,则失去极体的可能性更大。
  7. 将少量(约 2 - 3 µL)水吸入移液管。
  8. 用体视显微镜定位皮氏培养皿中的极体。
  9. 轻轻将极体吸到移液管中,并将其转移到含水悬滴载玻片上。轻轻吹送,将极体从移液管中释放出来,将其置于悬滴载玻片的中心圆圈内。
  10. 通过将极体与少量水一起吸入细长的移液管,将极体从水中移出。用少量水将细胞转移到显微镜载玻片上。在载玻片上水滴周围用碳化物记号笔刻画一个圆圈。
  11. 让极体在载玻片上的水滴中干燥,同时在倒置显微镜下持续观察。干燥后,将少量固定剂(约 2 - 3 µL)吸入同一细长的移液管并将固定剂滴到极体上。

    注意:当从取回起 6 小时后固定第一极体时,在即将完全干燥之前滴下固定剂以除去所有细胞质。
  12. 除非已经发生明显的细胞质溶解,在完全干燥之前,从细长的移液管中将另一滴固定剂置于细胞上。重复上述步骤,直到细胞周围的细胞质溶解,仅留下细胞核(使用固定剂至少 5 次)。

    注意:完全去除细胞质对于最佳探针杂交是必不可少的。
  13. 用划线器在玻璃载玻片顶部染色质周围画两个圆圈。一个圆圈比另一个圆圈稍大,即一个圆圈在另一个圆圈内,而细胞在较小圆圈的中间。刻画在细胞周围的圆圈将使杂交过程之后的细胞定位更加容易。
  14. 将载玻片放入装有甲醇的 Coplin 染色缸中 5 分钟。
  15. 将玻片从甲醇中取出并风干。
  16. 使用显微镜载物台上的游标标尺或 England Finder,在显微镜载玻片上定位并记录细胞位置。这些坐标将有助于杂交后细胞的定位。
  17. 在显微镜载玻片底部用永久标记记号笔在细胞周围画出另一个小圆圈。以此标明将要应用探针的区域。

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