Fluorescent immunohistochemistry was performed as previously described (Mommersteeg et al., 2010). Embryos were dissected and fixed overnight in 4% paraformaldehyde (ChemCruz, SC-281692). Following a brief wash in PBS (Gibco, 18912–014), the samples were dehydrated using an ethanol gradient (2 hr incubations in 70%, 80%, 90%, and 96% and two 1 hr incubations in 100% ethanol, Sigma-Aldrich, 32221–2.5 L-M) before being incubated in 1-butanol (MP Biomedicals, 194001) at room temperature overnight. Before mounting the embryos in sectioning moulds, three 2 hr incubations were performed in paraffin (Paraplast plus, Sigma-Aldrich, P3683) at 65 °C. Using a Leica microtome (Microm HM325), 10 μm sections were collected, mounted on Superfrost Plus glass slides (VWR, 631–0108) ensuring even coverage of the heart (one in every three sections per staining combination) and were allowed to dry overnight at 33 °C.
The slides were de-waxed in Xylene (Fisher Scientific, X/0200/17) twice for 5 min and rehydrated in an ethanol gradient (100%, 100%, 96%, 90%, 80%, 70%) for 1 min each before being incubated for one more minute in PBS. The samples were then boiled for 4 min in a pressure cooker in antigen-unmasking solution (H-3300, Vector Laboratories) and were allowed to cool down in PBS-T (Tween-20 0.1%, Chemcruz, sc-29113) before a ring was drawn around them using an ImmEdge pen (Vector Laboratories, H4000). The sections were blocked for 30 min at room temperature in a humidified chamber using blocking reagent [0.5% blocking reagent powder (Akoya Bioscience, FP1012), 0.15 M NaCl, 0.1 M Tris-HCl (pH 7.5)] and were then incubated with primary antibody diluted in blocking solution overnight at room temperature. Following three 5 min washes in PBS-T, the secondary antibody was applied (1:200 in blocking solution, Invitrogen, Alexa range) for 2 hr at room temperature.
For the Slit and Robo antibodies, an additional amplification step was added to enhance the signal using the TSA kit (NEL756001KT, Perkin and Elmer). Instead of an Alexa secondary antibody, a biotinylated secondary antibody was used at a 1:200 dilution in blocking solution for 45 min at room temperature, followed by three 5 min washes in PBS-T prior to a 30 min incubation with conjugated Streptavidin-Horse Radish Peroxidase (Vector Laboratories, SA-5004) and then three 5 min washes in PBS-T. Either fluorescein or tetramethylrhodamine (in DMSO, NEL756001KT) diluted at 1:100 in amplification buffer was then added to the sections for 3 min, followed by three 5 min washes with PBS and staining with DAPI (2.5 µg/ml, Sigma, MBD0015). Mowiol 4–88 (Applichem, A9011,1000) was used to mount the slides, which were cured at 37 °C in the dark. Primary antibodies against the following proteins were used: goat polyclonal anti-cardiac Troponin I (cTnI, 1:200; Hytest Ltd, 4T21/2), mouse monoclonal anti-Myosin heavy chain (MF20, 1:50, HSHB, AB-2147781), rabbit polyclonal anti-Peripherin (Prph, 1:200; Millipore, AB1530), rabbit polyclonal anti-Tyrosine Hydroxylase (TH, 1:200; Millipore, AB152), goat polyclonal anti-Robo1 (1:200; R&D Systems, AF1749), goat polyclonal anti-Robo2 (1:200; R&D Systems, AF3147), sheep polyclonal anti-Slit2 (1:200; R&D Systems, AF5444), rat monoclonal anti-Slit3 (1:200; R&D Systems, MAB3629), rabbit polyclonal anti-Erg1 (1:200, Abcam, ab110639), and rabbit polyclonal anti-Smooth muscle myosin heavy chain 11 (Myh11, 1:200, Abcam, ab125884). Imaging was performed on a Nikon microscope (Nikon, Eclipse Ci-L). Images were processed using ImageJ to generate magenta and green colour combinations (Schneider et al., 2012).