Sagittal sections of 5 days post-fertilization drl:APEX2-mCherry+ zebrafish larva in the region of the anterior kidney (eLife Table 1: APEX2 #1 5 dpf)
Larvae preparation for APEX2+;mCherry+ CLEM (eLife: Workflow #1). 5 dpf zebrafish larvae were prepared for MicroCT and SBEM as previously described (Deerinck et al., 2010). Briefly, immediately after light sheet imaging, larvae were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.15 M cacodylate buffer (CB, pH 7.4) at 4°C overnight. After removing the fixative, larvae were treated for 15 minutes with 20 mM glycine in 0.15M CB, on ice to quench the unreacted glutaraldehyde. Preincubation was done in 2.5 mM DAB solution (25.24 mM stock in 0.1 M HCl) in 0.15 M CB for 1 hour on ice. For staining, 0.03% H2O2 containing DAB solution was added to the larvae on ice. The reaction was monitored every 5-10 minutes and stopped when the desired intensity was achieved. The staining buffer was washed off with 5x washes using 0.15 M CB. Then, larvae were washed with 0.15 M CB and then placed into 2% OsO4/1.5% potassium ferrocyanide in 0.15 M CB containing 2 mM CaCl2. The larvae were left for 30 min on ice and then 30 min at room temperature (RT). After thorough washing in double distilled water (ddH2O), larvae were placed into 0.05% thiocarbohydrazide for 30 min. Larvae were again washed and then stained with 2% aqueous OsO4 for 30 min. Larvae were washed and then placed into 2% aqueous uranyl acetate overnight at 4°C. Larvae were washed with ddH2O at RT and then stained with 0.05% en bloc lead aspartate for 30 min at 60°C. Larvae were washed with ddH2O and then dehydrated on ice in 50%, 70%, 90%, 100%, 100% ethanol solutions for 10 min at each step. Larvae were then washed twice with dry acetone and placed into 50:50 Durcupan ACM:acetone overnight. Larvae were transferred to 100% Durcupan resin overnight. Larvae were then flat embedded between glass slides coated with mould-release compound and left in an oven at 60°C for 72 h. The larva block was imaged with a Merlin scanning electron microscope equipped with a 3View2XP and OnPoint backscatter detector. The volume was collected at 3 kV, with 10.8 nm pixels x and y; 70 nm Z steps and 70% local gas injection. The raster size was 25k x 12k and the volume was aligned using cross correlation and visualized using IMOD.