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H 1 glycine in PBS and blocked with 2 bovine serum albumin (BSA) PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22026160 in PBS at PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20460822 room temperature for one hour. Primary antibodies were applied with 1 BSA in PBS overnight at 4 and then rinsed twice with PBS containing 0.2 Tween-20. Secondary antibodies were applied with 1 BSA in PBS at room temperature for one hour and then slides were rinsed with PBS containing 0.2 Tween-20 and s40337-016-0117-z PBS. Coverslips were applied to slides after application of Vectashield?mounting solution with 4,6-diamidino-2-phenylindole (DAPI) (1:1 H-1000 to H-1200, Vector Laboratories, Inc., Burlingame, CA, USA) used as a nuclear marker. Negative control samples were incubated identically, except withoutTable 1 Sequence information for primers used in RT-qPCRGene symbol Actb Bgn Col1a1 Col3a1 Col5a1 Col11a1 Col12a1 Col14a1 Dcn Emcn Scx Tnmd Forward primer sequenceThese studies utilized both general distribution descriptions as well as tests of statistical significance. Features Methyl 2-((4-nitro-1h-pyrazol-1-yl)methyl)benzoate of the fibril diameter distributions for each construct group included mean fibril diameters, standard deviations, median diameter, and first and third quartile values. Statistical analyses of fibril diameter distributions were performed using the unpaired two-tailed t-test comparing mean fibril diameters of each image analyzed for each group (E15-17, TP, TP + GDF5, PERI, PERI + GDF5). RT-qPCR results for progenitor-seeded construct profiles and for expression profiling results from the conditioned media study were analyzed using non-parametric Mann hitney-Wilcoxon tests on unpaired samples.ResultsUltrastructural analysis of progenitor-derived matrix assemblyThis series of experiments tested the hypothesis that the tendon proper and peritenon progenitors have unique tenogenic properties when incorporated into the in vitro regenerative model. The structures of the engineered tissues and the developing tendons were analyzed using transmission electron microscopy. The progenitor-seededReverse primer sequence 5-CACAGCCTGGATGGCTACGT-3 5-ACTTTGCGGATACGGTTGTC-3 5-AGGAAGCTGAAGTCATAACCGCCA-3 5-TGGGGTTTCAGAGAGTTTGG-3 5-AGCAGTTGTAGGTGACGTTCTGGT-3 5-AGCCCTTGAGACCTCTGACA-3 5-AAGCGACGCAGAGAAAACAT-3 5-AGGCCAGTCAGAGCATCACT-3 5-AAGTCATTTTGCCCAACTGC-3 5-ACAGAGGCTTTTGTTGTGGAAGTT-3 5-TGTGGACCCTCCTCCTTCTAAC-3 5-CCAGCATTGGGTCAAATTCA-5-AGATGACCCAGATCATGTTTGAGA-3 5-CTACGCCCTGGTCTTGGTAA-3 5-TTCTCCTGGCAAAGACGGACTCAA-3 5-CACGCAAGGCAATGAGACTA-3 5-AAGCGTGGGAAACTGCTCTCCTAT-3 5-CTGGTCATCCTGGGAAAGAA-3 5-TGACTACGGTGCAGATGAGC-3 5-ACCTGTGAGTGTCCCTGGTC-3 5-TGAGCTTCAACAGCATCACC-3 5-CCAACAGTCTCTGCCACAGTGA-3 5-AAGTTGAGCAAAGACCGTGACA-3 5-CGCCACACCAGACAAGCA-Mienaltowski et al. Stem Cell Research Therapy 2014, 5:86 http://stemcellres.com/content/5/4/Page 5 ofFigure 2 Progenitor-derived matrix assembly is ultrastructurally similar to that of embryonic tendon. Ultrastructure was examined by cross-sectional images. In both the E15 to E17 Achilles tendon (A) and the progenitor constructs (B-E), collagen fibril synthesis and fiber assembly by cell processes are evident with fibers labeled with black dashed-line ovals (A-E). Panels: Embryonic Achilles tendon (A), tendon proper (TP)-derived progenitor-seeded construct (B), peritenon (PERI)-derived progenitor-seeded construct (C),TP-derived progenitor-seeded construct 5-(2-Fluorophenyl)-1H-pyrrole-3-carbonitrile supplemented with GDF5 (D), peritenon (PERI)-derived progenitor-seeded construct supplemented with GDF5 (E). ETC: embryonic tendon cell; TP: tendon proper-derived progenitor; PP: peritenon-derived progenitor (Bar: 1 m). E, embryo.