We also found that Prdm1 was expressed in the nuclei of all somitic myocytes. We found this same pattern of Prdm1 nuclear immunostaining with three different antibodies that were prepared against two different epitopes that are conserved between human and chicken Prdm1. Prdm1 expression was not limited to differentiated myocytes in the somite cultures, as most MyHC-negative cells in these cultures also showed nuclear staining for Prdm1. We also found that Prdm1 was expressed in the different fast and fast/slow types of MyHC-expressing cells that formed in cultures of E4 hindlimb and forelimb bud cells. As found previously, the differentiated muscle cells formed in E4 limb bud cultures after 2�C4 days of differentiation were small. Also consistent with previous studies, about two-thirds of the differentiated cells were of the fast type that reacted with mAb F59 but not mAb S58 and thus expressed only fast MyHC, whereas the remaining differentiated cells were of the fast/slow type that reacted with both mAbs F59 and S58 and thus co-expressed both fast and slow MyHCs. A previous study had shown that the fast/slow type of differentiated cells also express the slow MyHC1 isoform in addition to the S58-reactive slow MyHC2/3, but that none of the three slow MyHCs is expressed in the fast type of embryonic myotubes in culture. By immunostaining, we found that Prdm1 was expressed in the nuclei of both the fast and fast/slow types of differentiated cells in the E4 limb cultures. Prdm1 was also expressed in most of the cells that did not express MyHC. These results, which were obtained with both the Cell Signal Pteryxin Technology and Abcam antibodies, showed that Prdm1 expression in differentiated E4 limb cells was found in cells that expressed only fast MyHC, as well as in those that co-expressed fast and slow MyHC. We also found that Prdm1 was expressed in the myotubes formed from fetal E12 limb myoblasts. As in previous work, we found that fetal E12 myoblasts formed very large, multinucleate myotubes that reacted with mAb F59 but did not react with mAb S58 after 3�C4 days in differentiation NVP-BAG956 medium.