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Assays, 50 isolated cells were replated in {each
Dox (Sigma-Aldrich) was added to hanging drops at corresponding days to a final concentration of 1 /ml as previously described (Bondue et al., 2008). Immunofluorescence analysis Fixation, blocking, and principal and secondary antibodies as well as mounting medium employed within this study were previously described (Bondue et al., 2008), except for the anti-GFP staining (rabbit polyclonal; 1:1,500; Invitrogen).Counterstaining of purchase JK184 nuclei was performed with Hoechst (1:two,000; Invitrogen). Immunostaining was acquired making use of a microscope (Axio Observer.Z1), a camera (AxioCam MR3 or MRc5), as well as the Axiovision application (Carl Zeiss, Inc.). Acquisitions have been performed at space temperature employing ten and 20EC Program Neofluar objectives (10= 0.3 numerical aperture and 20= 0.four numerical aperture; Carl Zeiss, Inc.). Mosaics were generated by the Axiovision software program utilizing a 10 overlap among every single single acquisition. Confocal pictures were acquired at space temperature employing a multiphoton confocal microscope (LSM510 NLO; Carl Zeiss, Inc.) fitted on an inverted microscope (Axiovert M200; Carl Zeiss, Inc.) equipped with C-Apochromat (40= 1.2 numerical aperture and 63= 1.2 numerical aperture) water immersion objectives (Carl Zeiss, Inc.). 0.35-mm-thick, 512 512 ixel optical sections had been collected sequentially for each and every fluorochrome. The datasets generated were merged and displayed using the LSM510 software program and exported in TIF image format. Live-sample imaging 8 d immediately after cell isolation at D3 and replating on gelatin-coated dishes, beating regions have been imaged by time-lapse bright-field acquisitions applying a microscope (Axio Observer.Z1) and the Axiovision computer software. All acquisitions had been performed at area temperature utilizing a 10EC Strategy Neofluar objective (0.three numerical aperture). Image sequences have been compiled together with the Axiovision application, and video files show 15 images/s. RNA isolation, reverse transcription, quantitative PCR, and single-cell PCR RNA extraction, DNase treatment, and RT-PCR had been performed as previously described (Bondue et al., 2008). Quantitative PCR was performed making use of Brilliant II Fast SYBR qPCR Master Mix (Agilent Technologies) on a real-time PCR technique (Mx3005P; Agilent Technologies). All primers have been designed working with Lasergene 7.two software (DNAStar, Inc.) and are listed in Table S2. Single-cell PCR, generation of cDNA, and PCR amplification were performed as previously described (Jensen and Watt, 2006). In short, just after cDNA synthesis, two rounds of 35 cycles of amplifications have been performed by PCR, as well as the amplification product was employed as a PCR template for the detection of gene expression. For single-cell PCR experiments, CXCR4/PDGFRa/Flk1 TP cells had been sorted straight in 96-well plates containing the very first strand buffer. The cDNA amplification process was utilised for the expression profiling of colonies obtained immediately after differentiation of single Mesp1-GFP cells sorted in 96 wells.Assays, 50 isolated cells were replated in each and every well of an 8-well Lab-Tek glass chamber slide (Thermo Fisher Scientific) with Y-27632 (EMD) at a final concentration of 10 for the very first 48 h. Dox-inducible ESC lines had been differentiated in DME containing 15 ESC-qualified serum and 0.5 mM ascorbic acid (Sigma-Aldrich). Following four d in hanging drops, ESCs have been replated on gelatin-coated dishes for further differentiation.
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