A1r multiphoton upright confocal microscope

Embryos were fixed for 1 hour at room temperature in 1% formaldehyde in PBS in 3 ml glass vials. Embryos were washed 1X in PBS and then 2X in 0.2% saponin/1X PBS, followed by blocking in 0.2% saponin/0.5%sheep serum/1X PBS (Saponin blocking solution) for one hour. AMHC (S46) and MHC (sarcomeric myosin; MF20) primary antibodies (Developmental Studies Hybridoma Bank) were incubated at 1:10 in Saponin blocking solution. Rabbit polyclonal DsRed antibody (Clontech), to detect mCherry, and Living colors anti-RCFP (Clontech), to detect ZsYellow, were used at a 1:1000 dilution. Rabbit anti-GFP (Abcam) was used at 1:500. Rabbit anti-Nkx2.5 (Gene Tex) was used at 1:250. Mouse anti-pHH3 (Abcam) was used at 1:1000. All secondary antibodies were used at dilutions of 1:100. Antibody information is also listed in S2 Table. Cell counts were performed by gently flattening embryos under a coverslip and counting the fluorescent nuclei in each chamber. For all imaging except Nkx2.5/pHH3, embryos were imaged using a Zeiss M2BioV12 Stereo microscope. For Nkx2.5/pHH3, embryos were post-fixed in 2% formaldehyde/1X PBS for two hours and mounted in 1% low-melt agar on 2% agar plates. Images of one side of the embryo were taken using a Nikon A1R Multiphoton Upright Confocal Microscope with a 16X water immersion objective. 200 μm optical sections were taken with the resonance scanner.