Planapo 20x 0.8 na objective

Tg(myl7:Twitch-4) larvae at 3 dpf were euthanized by incubation in 0.3% MS-222 (Sigma-Aldrich A5040) for 5 min. Larvae were embedded in agarose as done for ratiometric imaging and were imaged in an inverted Axio Observer LSM710 confocal microscope (Carl Zeiss, Germany) with a PlanApo 20x/0.8 NA objective. Laser excitation was at 488 nm and the emission bandwidth was 520-560 nm.

At the specified day of differentiation, cell media was removed, and coverslips were treated with 4% PFA for 15 min at RT, followed by three washes with 1xPBS and permeabilization in 0.1% Triton X-100 in PBS for 3 min at RT. Prior to staining, cells were incubated in a blocking solution of 3% BSA in PBS for 20 min at RT. Then, primary antibodies (rat anti-MBP and/or rabbit anti-RFP) were added in a 3% BSA solution for overnight incubation at 4 C. On the following day, the primary antibody solution was rinsed off with three washes of PBS, and then incubated with secondary antibodies (anti-rat AlexaFluor 594 or 647) in 3% BSA for 1 hr at RT. After three washes with PBS, CellMask Blue stain (1:1000) and phalloidin-488 (7 μL phalloidin per 1 mL PBS) was incubated to stain all cells for 15 min at RT, followed by three additional rounds of washing with PBS. Stained cells were mounted onto microscope slides (Fisher Scientific 12-550-143) in Fluoromount G (SouthernBioTech, 0100-20).
Cells were imaged by widefield epifluorescence with a Zeiss Axio Observer Z1 using the Plan-Apo 20x/0.8 NA objective for actin, MBP, and cell area quantifications. Images were acquired blinded to the genotype or condition with identical illumination and acquisition conditions per biological replicate.