免疫荧光染色固定问题

wxyWXY

请教一下  想做小鼠ips得多能性基因免疫荧光染色 细胞用4％pfa固定15分钟后 用pbs 洗一遍  然后放在4度过夜  第二天再染色时发现没有细胞了  是不是pbs中放的时间太长了 细胞再染色时就被冲走了 准备了好长时间得细胞 全没有了 :'(

xiaomingxu

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# h7 T  d, [# }: ]% U' c6 \细胞经多聚甲醛固定后，PBS中4度过夜应该是没有太大影响的，至于你出现的问题可能的解释是细胞贴壁不牢，经过你多次清洗操作后有可能漂浮起来，但是没有关系，你可以通过在倒置显微镜下观察在悬液中找回来；或者是操作太过激烈，再固定前就已经将细胞冲走了。以后还是按照操作规程来做啦，继续加油！以下是细胞免疫荧光的基本程序：4 O6 A$ }. \4 T
Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (18–22 °C) and permeabilized in 0.05% Triton-X100 in PBS for 10 min at room temperature, then blocked in 1% BSA in PBS for 1 h at room temperature. Cells were washed with PBS and incubated with antibody to Nanog (1:300, rabbit polyclonal; ab21603, Abcam) and/or antibody to Oct4 (1:50, mouse monoclonal, C-10; Santa Cruz) over night at 4 °C. After washing with PBS (6 × 10 min), cells were labeled with Alexa 488– or Alexa 555–conjugated secondary antibodies (Invitrogen) for 1 h at room temperature. Cells were then washed with PBS (3 × 10 min) and nuclei were counterstained with 10 ug/ml DAPI in PBS for 8 min at room temperature. After final washing with PBS (3 × 10 min), specimens were analyzed using a fluorescence microscope or Radiance2000 confocal microscope (Bio-Rad).

aminhair

:)你操作时吸力是不是过大了?尤其是洗的时候！

wxyWXY

谢谢大家啊 :D

大刀无敌

pbs使用时建议平衡至常温再用，可减少细胞的漂浮现象。millipore 有一种专用染色的培养新产品，可以尝试一下。