Immunofluorescence

Whole-mount double immunofluorescence was conducted using a protocol described previously (Alexander et al., 1998) for the following primary antibodies: anti-Islet1/2 (1:50; 39.4D5, Developmental Studies Hybridoma Bank), anti-atrial myosin heavy chain (1:20; S46, Developmental Studies Hybridoma Bank), anti-RCFP (1:50; Clontech, 632475) and anti-GFP (1:100; Life Technologies, A11122). When employing the following primary antibodies, anti-Eln2 (1:500; Miao et al., 2007) and anti-sarcomeric myosin heavy chain (1:20; MF20, Developmental Studies Hybridoma Bank), another published protocol was used (Zhou et al., 2011). The following secondary antibodies were employed: goat anti-mouse IgG2b Alexa Fluor 488 or 568, goat anti-mouse IgG1 Alexa Fluor 488 or 568, goat anti-mouse IgG2a Alexa Fluor 488, goat anti-rabbit IgG Alexa Fluor 488 or 568, and goat anti-mouse IgG Cy5 (all at 1:200; Invitrogen).