4.6. Scanning Electron Microscopy (SEM) for IHC-CLEM

After CLSM acquisition, sections were contrasted according to Reynolds [55], attached to double-sided tape mounted on microscopy glass slides with sections facing up and platinum-coated with a MC1000 sputter coater (Hitachi) for 20 s at 10 mA. Observation by SEM of the regions of interest were achieved on a SU5000 scanning electron microscope (Hitachi High-Technologies, Tokyo, Japan) operated in high vacuum at 7 keV with BSE detector and observation mode (spot size 30). Working distance was 6 mm. Capture speed was 40 s. Image size was 1280 × 960 pixels. Magnification ranged between ×180 and ×60,000, with pixel size between 551 and 1.6 nm/pixels, respectively.