Immunohistochemical Analysis of Vestigial Protein

For the immunohistochemical analyses the material was fixed in 4% formaldehyde. Samples were dehydrated in series of ethanol and HistoChoice^® Clearing Agent (Sigma-Aldrich) and embedded in paraplast. The blocks were cut into 1 to 5-μm-thick sections. Slide-mounted sections were dewaxed in HistoChoice^® Clearing Agent (Sigma-Aldrich), rehydrated gradually through a series of ethanol dilutions and rinsed in water (for further details see^{10 (link)}). Blocking of non-specific binding sites was performed with casein blocking buffer (Thermo Fisher) overnight at 4 °C prior to the incubation with the rabbit anti-Vestigial antibody diluted 1:500. In parallel performed control experiments, the primary antibody was omitted. After overnight incubation at 4 °C in humid chamber, Cy3 goat anti-rabbit secondary antibodies (Life Technologies) were used. Incubation with secondary antibodies was carried out for 4 h at room temperature. After rinsing with PBS, the sections were mounted in ProLong Gold antifade reagents with DAPI (Invitrogen) and analyzed in the DMR Leica epifluorescence microscope (FM) equipped with appropriate filters.