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[lucy68]

Does anybody have a good protocol for staining mouse ES clones for confocal analysis? I have two problems
1. I use 2 Ab one works only with MetOH fixation but the other that is nuclear (transcription factor) with metOH looks fuzy,
2. the antibodies don't reach the cells inside the clone although I have used triton 0.5% for 10 min
please any suggestion would be appreciated! :(  :(  :(

[cellcounter]

lucy68, on Jun 25 2009, 03:06 AM, said:

Does anybody have a good protocol for staining mouse ES clones for confocal analysis? I have two problems
1. I use 2 Ab one works only with MetOH fixation but the other that is nuclear (transcription factor) with metOH looks fuzy,
2. the antibodies don't reach the cells inside the clone although I have used triton 0.5% for 10 min
please any suggestion would be appreciated!     

1. I would try out Paraformaldehyde fixation, preferably with Sucrose.


2. ES clones are thick affair, minute cells grow up in heaped up fashion, and as you suggest, your tritonx-100 may not be reaching the cells buried in the heap, which is what are visualized in confocal z-scan. So, unless you dissociate these clones to make a kind of monolayer, or do regular IF microscopy, you may not see the signal.

HTH/