Anti ctni

After 1 and 2 weeks of culture, cellular functions including cardiomyogenesis and angiogenesis were assessed using an immunofluorescence method. After the predetermined period, the cell-laden constructs were fixed with formalin for 20 min. The samples were permeabilized in 0.1% Triton X-100 for 15 min and were then incubated with a blocking solution [containing 1% bovine serum albumin, 0.1% Tween 20, and 0.3 M glycine in PBS] for 2 hours. The cells were then incubated with primary antibodies at 4°C overnight. After incubation with primary antibodies, secondary antibodies were introduced to the samples in the dark for 2 hours at room temperature, followed by incubation with a DAPI (1:1000) solution for 5 min. All images were obtained using the confocal microscope, and protein quantifications were performed using ImageJ (49 ). In addition, the immunostaining analysis was performed with sliced fragments that were cut with a cryostat microtome. The primary antibodies that were used for our study were purchased from Abcam and included anti–α-actinin (1:500), anti–human-specific CD31 (human-specific PECAM-1; 1:500), anti-desmin (1:1000), anti-Cx43 (1:1000), anti-cTnI (1:500), and anti-vWf (1:1000). The secondary antibodies were purchased from Thermo Fisher Scientific and included anti-mouse Alexa Fluor 594 (1:1000) and goat anti-rabbit Alexa Fluor 488 (1:1000).