Discovery microscope

Animals were mounted ventral up in Vectashield mounting medium. Fluorescent images were taken with a Zeiss LSM700 Confocal Microscope using ZEN 2011 software with following objectives. For transverse cryosection shown in Fig. 4e and Supplementary Fig. 9b, stained animals were embedded in Tissue-Tek O.C.T. Compound (Electron Microscopy Services) and sectioned at 25 μm. All images and quantification for co-expression and for determining cell localization relative to muscle fiber layer were analyzed at 40X NA 0.75 with minimum 0.31 × 0.31 × 1.11 μm resolution or 63X NA 1.4 with 0.2 × 0.2 × 0.57 μm resolution with the exception of H3P⁺smedwi-1⁺ (Fig. 4d) which was quantified at 20X NA 0.8 with minimum 0.63 × 0.63 × 2.04 μm resolution. Animal overview images were acquired with 10X NA 1.45 with minimum 1.25 × 1.25 × 7.26 μm resolution. Linear adjustments of brightness and contrast equally across controls and experimental images, re-assignment of linear look-up table colors, co-localization analysis of FISH signals, and maximum intensity projections through area of interest was performed using Fiji with ImageJ 2.0.0^{76 (link)}. Live animal images were taken with a Zeiss Discovery Microscope using AxioVision v4.7.1.

WISH with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) was performed as described (Pearson et al., 2009 (link)). FISH and post-antibody binding washes and tyramide development were performed as described (King and Newmark, 2013 (link)). Briefly, animals were killed in 5% NAC and treated with proteinase K (2 μg/ml). Animals were hybridized with RNA probes at 1:800 dilution overnight at 56°C. Samples were then washed twice in pre-hybridization buffer, 1:1 pre-hybridization: 2X SSC, 2X SSC, 0.2X SSC, and PBS with Triton-X (PBST) in that sequence. Blocking was performed in 5% Western Blocking Reagent (Roche) plus 5% heat-inactivated horse serum diluted in PBST solution when anti-DIG, anti-FITC, or anti-DNP antibodies were used. Post-antibody binding washes and tyramide development were performed as described (King and Newmark, 2013 (link)). Light images were taken with a Zeiss Discovery Microscope. Fluorescent images were taken with a Zeiss LSM700 Confocal Microscope. Fiji/ImageJ (RRID: SCR_002285) was used for FISH co-localization analyses and PCG domain quantifications.

Animals were wounded and fixed at six hours following injury in 4% formaldehyde [50] (link) and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) colorimetric whole-mount in situ hybridizations or fluorescence in situ hybridizations (FISH) were performed as described [50] (link). For immunostainings, animals were fixed as for in situ hybridizations and then treated as described [51] (link). A mouse anti-ARRESTIN antibody was kindly provided by Kiyokazu Agata and used in a 1∶5,000 dilution, and an anti-mouse-Alexa conjugated antibody was used in a 1∶500 dilution. For the neoblast wound response assay, RNAi animals were fed during the course of eight weeks every three to four days, then were transversely amputated and trunk or tail fragments were fixed at 0, 6, 18, 48, and 120 hours following wounding. Animals were immunostained using a rabbit anti-phospho histone 3 antibody and an anti-rabbit HRP in a 1∶100 dilution as previously described [52] (link). Fluorescent images were taken with a Zeiss LSM700 Confocal Microscope. Light images were taken with a Zeiss Discovery Microscope.

Fluorescent images were taken with a Zeiss LSM700 Confocal Microscope. All images are Maximum intensity projections unless otherwise indicated in figure legends. Light images were taken with a Zeiss Discovery Microscope.

To visualize the coronary vessels in embryonic hearts, whole-mount staining was performed using antibodies against PECAM1, EMCN or LYVE1 as described previously^{64 (link)}. Briefly, the hearts were isolated from E16.5 control and Pofut1^cKO embryos, and fixed in 4% PFA for 2–3 h at 4 °C. The hearts were then blocked in 5% normal horse serum for 3 h at 4 °C and followed by incubation with primary antibodies at 4 °C overnight. The hearts were then washed for five times with PBS containing 0.5% TritonX-100 and incubated with biotinylated secondary antibodies at 4 °C overnight. The hearts were then washed for five times with PBS containing 0.5% TritonX-100 and incubated with streptavidin from ABC Kit (Vector Laboratories) at 4 °C overnight. The hearts were then washed for five times with PBS containing 0.5% TritonX-100 and developed color using DAB Kit (Vector Laboratories). In addition, coronary angiogram was performed to access the embryonic coronary arteries by injecting fluorescence dye into the left ventricle of beating hearts. The dye entered and labeled the coronary arteries through the coronary ostia (the coronary openings at the root of aorta). After injection the hearts were collected, fixed in 4% PFA, and photographed using a Zeiss Discovery Microscope.