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      Immunofluorescence Microscopy and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Including Primary Cilia Associated Signaling Pathways.

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          Abstract

          This chapter describes the procedures for immunofluorescence microscopy (IFM) and quantitative PCR (qPCR) analyses of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol is provided outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IFM and qPCR anlysis. The techniques are illustrated with results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, PDGF, and TGFβ signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment is experimentally shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.

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          Author and article information

          Journal
          Methods Mol. Biol.
          Methods in molecular biology (Clifton, N.J.)
          Springer Nature America, Inc
          1940-6029
          1064-3745
          2016
          : 1307
          Affiliations
          [1 ] Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen, Copenhagen OE, Denmark, maj.linea.vestergaard@rh.regionh.dk.
          Article
          10.1007/7651_2014_127
          25304206

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