Dear all, I'm in the middle of characterising a couple of teratocarcinoma derived progenitor cell lines namely mouse P19 and human NTERA2/D1. I would really like to quantify the expression of an embryonal carcinoma associated transcription factor - Oct 4 (octamer binding protein 4) by flow cytometry. To date I have had success with immunohistochemical localisation using a BD pharmingen anti-oct 3/4 antibody (cat no 611202) and either HRP or FITC conjugated secondaries. The immunohistochemical protocol works well (nice strong nuclear localisation) simple using formal saline fixed cell monolayers and 0.1% triton in the wash buffers. However if I try to repeat the protocol on cells fixed in suspension and analysed by flow I cannot find any 'bright' cells. Any suggestions on why the adherent cell assay works well and the fixed cell flow assay does not would be appreciated. Many thanks Stewart
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