Histology and TEM Fixation Protocol

For histology and TEM, heads of 5 dpf larvae were fixed in 2% glutaraldehyde, 2% paraformaldehyde in 50 mM Hepes (pH 7.25) for 1.5 h at room temperature (RT), then at 4°C overnight. Heads were washed 5×3 min in Hepes buffer, 2×5 min in PBS, incubated for 60 min in 1% OsO₄/1.5% potassium ferrocyanide in PBS, washed 2×5 min in distilled water and dehydrated in an ethanol series. Samples were incubated 2×10 min in propidium oxide and embedded in Durcupan. For histology, semithin sections of 500 nm were stained with 1% Toluidine Blue, 0.5% sodium borate solution and mounted in Entellan (Merck). Samples were imaged with Zeiss Imager.Z1 microscope using AxioCam HRc and AxioVision software (Zeiss). For TEM, ultrathin sections of 70 nm were stained with uranyl acetate and lead citrate, and imaged with a Morgagni TEM (80 kV) using a Morada CCD camera (EMSIS GmbH) and ITEM software (EMSIS GmbH). Images were processed using TrakEM2 in Fiji (Cardona et al., 2012 (link); Schindelin et al., 2012 (link)) and Adobe Photoshop.