Mouse anti mf20

GelMA-myoblast constructs were fixed with 10% formalin for 30 min, then blocked and permeabilized for an hour with 10% normal donkey serum made up with a PBS of 0.1% TritonX-100. Immunofluorescent staining was performed for sarcomeric myosin (mouse anti-MF20, Developmental Studies Hybridoma Bank). Cells were incubated in the primary antibody (1:400) overnight at 4 °C. Cells were then incubated with the secondary antibody Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:2000, Molecular Probes) and Alexa Fluor 488 Phalloidin (1:100, Thermo Fisher Scientific) for 60 min at 37 °C. Nuclei were stained with 1 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 15 min at room temperature. Samples were washed in PBS and imaged with an inverted fluorescence microscope (Olympus IX70). The 3D rendered z-stack images were taken with confocal microscopy. A total of 0.5 µm red fluorescent beads at a concentration of 25 µL/mL were added to the bioink (aqueous suspension of carboxylate–modified polystyrene latex beads, Sigma-Aldrich). After printing, the cells were then stained with Alexa Fluor 488 Phalloidin, as described above. Confocal imaging was performed with a NikonA1Plus confocal microscope using a Nikon Plan Fluor 20× DIC L N1 N.A. 0.75 objective lens, and the images were processed using NIS-Elements software (Nikon).

Flow cytometry analyses were performed as described previously (Zhang et al., 2012 (link)), with antibody concentrations as following: mouse anti-cTnT (1:200, Lab Vision), mouse anti-MF-20 (1:20 Developmental Studies Hybridoma Bank), and Alex Fluor 488 goat anti-mouse IgG (1:500, Life Technologies). Data were collected and analyzed on a LSRII flow cytometer (Becton-Dickinson).