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Cy3-labled cRNA (0.6?��g) was fragmented to an average size of 50�C100 nucleotides by incubation with fragmentation buffer at 60?��C for 30?min. The corresponding fragmented and labeled cRNA was then pooled and hybridized to Agilent SurePrint G3 Mouse GE 8?��?60?K Microarray (Agilent Technologies, USA) at 65?��C for 17?h. After washing and then blow drying with a nitrogen gun, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535?nm for Cy3. Scanned images were analyzed by Feature Extraction Software, version 10.5.1.1 (Agilent Technologies, USA). Representative cultures from 2D and 3D cultures were fixed in formalin, permeabilized with 0.1% Triton Fluvoxamine X-100, blocked in 10% BSA, and stained with anti-connexin 43 (1:200) rabbit polyclonal antibody (Cell Signaling Technologies, USA) followed by a goat anti-rabbit Alexa Fluor 488 (1:1000) antibody (Invitrogen, New York, USA). Samples were stained with Alexa Fluor 594-conjugated anti-Troponin I heavy chain (Santa Cruz Biotechnology, Dallas, USA); all scaffolds were counterstained with DAPI (Invitrogen, New York, USA) to visualize cell nuclei and imaged using a Zeiss LSM 510 multiphoton confocal microscope (Carl Zeiss I-BET-762 order Microscopy, New York, USA) or IX51 (Olympus). Primary neonatal mouse heart cell culture medium was measured by ELISA. Briefly, 96-well plates were coated with an anti-mouse B type natriuretic peptide (BNP) antibody (Abnova). The anti-BNP antibody was added and incubated at room temperature for 2?h. Claycomb culture medium was added and incubated for 2.5?h. A streptavidin-horseradish peroxidase (HRP) solution was added and incubated at room temperature for 45?min followed by addition of a substrate solution. The optical density in the wells was monitored by a microplate reader (BioTek, ��Quant Microplate Spectrophotometer) at a wavelength of 450?nm. The experiments were repeated 5 times to get the mean and for the statistical analysis. All continuous results were expressed as the mean?��?standard deviation (SD). Comparisons between groups for continuous Selleckchem Pazopanib data were made using Student��s t test. A p value of
