P8 hearts from virus injected pups were fixed in 10% NBF overnight, rinsed in PBS, dehydrated, and finally embedded in paraffin. Embedded specimens were then cut into 10 µm sections before mounting on glass slides and stored at 4 °C. Sections were deparaffinized and rehydrated before staining. Immunostainings were performed on NBF or PFA fixed cell cultures or paraffin embedded sections as previously described in Andersen et al. [2 (link)] with the following primary antibodies: rabbit anti-GFP (1:500, ab290, Abcam), mouse anti-MYH1 (1:300, MF20-c, DSHB), rabbit anti-ZEB1 (1:500, PA5-28,221, Thermo Fisher), rabbit anti-Mef-2c (1:500, 5030S, Cell Signaling Technology), mouse anti-actinin (1:200 A7811, Sigma), mouse anti-α-tubulin (1:250, 3873, Cell Signaling Technology), and 647-phalloidin (1:800, A30107, Thermo Fisher). Secondary antibodies used were: 488-donkey anti-rabbit (1:200, A21206, Invitrogen), 555-donkey anti-rabbit (1:200, A31570, Invitrogen), 647-donkey anti-mouse (1:200, A31571, Invitrogen). All sections were mounted with DAPI (Vectashield, Vector Lab., for Phalloidin and α-tubulin staining, Fluoroshield, Abcam was used). Microscopy was performed on a Leica DMI 4000 B microscope with a Leica CTR4000 illuminator and Leica DFC300FX/DFC 340 FX cameras, and confocal microscopy was performed on an Olympus FV1000MPE confocal laser scanning microscope equipped with an UPlanSApo 60x/1.20 water objective. During analysis, all camera settings and picture processing were applied equally to samples and controls.
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