Star 488

Cells were washed 2x gently with media, followed by a PBS++ (Ca2+ and Mg2+) wash. Then the cells fixed cells with 4% (v/v) PFA in PBS++ (Ca2+ and Mg2+) for 10–20 min at RT. Then cells were washed 3x with PBS. Cells were permeabilized using 0.01% Triton in PBS for 20 min at RT. Then the cells were washed 3x with PBS, and then blocked in 2% BSA for 20–30 min at RT. The primary antibody (anti-green fluorescent protein, REF A11122, Invitrogen by Thermo Fisher Scientific) was directly added the blocking buffer at 1:1000 dilution and incubated at 4 C overnight. The next day the cells were washed 3x with PBS, 10 min each. Then, the secondary antibody (Abberior STAR 488, Cat #2-0012-006-5, Abberior) was used at a dilution of 1:1000 and incubate for 2 hr at RT. The cells were washed 5x with PBS, 10 min each. Then the samples were mounted in Mowiol and stored at 4°C until imaging.

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).