Prolong diamond anti fade medium

Immunofluorescence examinations used thin myofiber bundles dissected from muscle biopsies. Bundles were mounted moderately stretched in relaxing solution, on Sylgard-coated dishes. Relaxing solution was replaced by fixative containing 4% PFA for 20 min. Bundles were transferred to a 24-well plate and washed three times for 10 min in PBS, then permeabilized with 0.1% Triton X-100 (Sigma) for 30 min at room temperature and blocked in 5% goat serum (Sigma) with slow agitation for 1 hr. The primary antibody was applied overnight at 4°C with agitation, followed by 3 PBS washes for 10 min. Fluorescent secondary antibody was applied for 2 hr at room temperature. Dehydrated bundles were mounted with Prolong Diamond anti-fade medium (Thermo-Fisher).

PHA that accumulated in the cells was stained with Nile red (Fujifilm Wako Pure Chemical, Osaka, Japan)^{14 (link)}. A glass slide was coated with 0.01 w/v% poly-l-lysine (Sigma-Aldrich, St. Lois, MO) for cell attachment. The culture suspension (100 µL) was placed on the poly-l-lysine-coated glass slide for 10 min and then aspirated. The cells attached to the glass slide were incubated with a drop of 10 µg/mL Nile red in D-PBS(−) (Nacalai Tesque, Kyoto, Japan) for 30 min, washed with D-PBS(−), mounted with Prolong Diamond antifade medium (Thermo Fisher Scientific, Waltham, MA) for immobilization, and covered with a coverslip. Cells were observed using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan). The frame of each individual cell was manually identified, and the cell area was calculated using the BZ-X800 Analyzer software (Keyence). PHA formation was visualized using a tetramethylrhodamine isothiocyanate filter set (λ_em ≈ 554 nm, λ_ex ≈ 570 nm). The fluorescence intensity of the Nile red-stained dot assembly was calculated on average using the BZ-X800 Analyzer software.

Isolation of embryonic cells was performed as described (Christensen et al., 2002 (link); Krieg et al., 2014 (link); Strange et al., 2007 (link)). We plated cells on glass bottom dishes (thickness #1.5, WillCo wells), culture medium was exchanged daily, and cells were analyzed cells after 3 to 7 days of growth in vitro. We prepared cells for immunofluorescence using a 5-min treatment with Krebs fixative containing 4% PFA (Electron Microscopy Sciences; vol/vol) at room temperature (RT) followed by a 15-min treatment in ice-cold methanol at −20°C. Next, we treated cells in antibody buffer (1xPBS +0.01% Triton X100 +3%BSA weight/vol) for 1 hr at RT, exposed them to a primary antibody raised against GFP (rabbit-αGFP; Life Technology) for 6 hr at RT with gentle agitation, and washed them three times in PBST for a 15 min. The primary antibody was diluted 1:100 from a stock solution (1 mg/ml) stored at −20°C, according to manufacturer’s instructions. We applied the secondary antibody (goat-anti-rabbit AbberiorStar 488, Abberior, 1:200 dilution) overnight at 4°C and washed for 4 hr at RT before mounting in Prolong Diamond antifade medium (LifeTechnology). Monoclonal antibody against acetylated tubulin was used at 1:2000 to identify TRNs in culture (SigmaAldrich, 6-11B, 6 hr at RT) and detected with Alexa-680 secondary antibody (goat-anti-mouse, Invitrogen, 1:2000, 4 hr at 4°C).

Cells were stained for immunocytochemistry and flow cytometry as described previously (Perriard et al., 2015 (link)). In brief, for immunocytochemistry, cells were fixed with 4% paraformaldehyde, blocked for 1 hr, then incubated overnight with primary antibodies. The next day, cells were incubated with secondary antibodies before counterstaining with DAPI and slides were mounted with ProLong Diamond Antifade medium (Life Technologies). For flow cytometry staining, cells were first stained with violet Live/Dead (Life Technologies) before staining for extracellular markers with labeled antibodies. For intracellular staining, cells were fixed and permeabilized before adding primary antibodies, then washed and stained with labeled secondary antibodies. See Supplemental Information for detailed methods (Table S2).