Efficient wet-spinning of pre-aligned microtissues for 3D bioprinting complex tissue alignment

To observe protein expression, wide-field epifluorescence was captured with an EVOS FL Auto 2 (Thermo Fischer) at days 0, 1, 3, and 7 during fiber development. Samples were fixed for 30 min with 4% paraformaldehyde (PFA) at room temperature, then washed 3X for 5 min with DPBS and used immediately or stored at 4 °C for later analysis. Blocking was performed for 1 h with 10% Horse Serum, 1% BSA. Primary anti-human antibodies were diluted in blocking buffer and incubated overnight as follows: Anti-SM-MHC (Proteintech, 21404-1-AP, rabbit polyclonal, dilution 1:100), anti-Calponin-1 (NeoBiotechnologies, 1264-MSM2-P1ABX, mouse monoclonal, dilution 1:200), anti-CX43 (Invitrogen, 13-8300, mouse monoclonal, dilution 1:100). Fibers were washed 3X for 5 min with DPBS before adding secondary antibodies diluted in 1% BSA and incubated at room temperature for 1 h. Secondary antibodies used were Cy3 conjugated with Cy3 goat-anti-mouse (Jackson ImmunoResearch Laboratories, 715-165-151, dilution 1:500) and goat-anti-rabbit DyLight 488 (Invitrogen, 35552, dilution 1:500). Fibers were then washed 3X for 5 min with DPBS. Some fibers were incubated in iFluor 488-conjugated phalloidin (Cayman Chemical, 20549) at 1:1000 dilution without other antibodies. Stained fibers were then rinsed 3X for 5 min in DPBS before mounting with DAPI mounting media and incubating for 1 h.