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Problem with permeabilisation specificity

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#1 charles_duhwin



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Posted 30 November 2010 - 04:11 PM


I am staining intact oocytes expressing a FLAG-tagged protein. I am trying to stain with and without permeabilisation, to demonstrate that the tag is in the predicted (intracellular) location. However, I seem to get equally good staining with or without permeabilisation using 0.1% Triton-X (15 min) after fixing with 2% PFA (4h at 4 degrees celcius). After fixation the oocytes are cryoprotected (48h) in 30% (w/v) sucrose-PBS, and I am wondering if anyone knows if this step could be permeabilising the cells. Thanks in advance if anyone can help.


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