Brief description of sample preparation
After cryofixation by high-pressure freezing and freeze-substitution, cryofixed antennae were rehydrated gradually. Rehydrated samples was then subjected to DAB labeling reaction and stained using a high-contrast en bloc staining protocol. Next, the samples were dehydrated for resin infiltration and embedding, followed by imaging with X-ray microscopy and then SBEM.

Serial Block-face Scanning Electron Microscopy
The Drosophila antenna was imaged using a Gemini scanning electron microscope equipped with a 3View2XP and OnPoint backscatter detector. Images were acquired at 2.5 kV accelerating voltage with a 30 μm condenser aperture and 1 μsec dwell time; Z step size was 50 nm; pixel size was 6.5 nm; raster size was 12k x 9k; Z dimension was 1200 sections, and local gas injection was set to 85%.Volume was aligned using cross correlation, segmented, and visualized using IMOD.