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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：3 u$ h! m& w" U! I5 B( H
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). 作者: aminhair 时间: 2011-3-22 12:20