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confocal anlysis on ES clones


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#1 lucy68

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Posted 25 June 2009 - 03:06 AM

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! :( :( :(

#2 cellcounter

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Posted 26 June 2009 - 08:15 AM

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! :huh: :) :(


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/




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