Placed in a silicon wafer, the region of interest (ROI) in the sections was identified by visualizing blood vessel land marks. Electron micrographs at higher resolution (4 nm pixel size, 1 µs dwell time) were obtained with a Sigma Field Emission Scanning Electron Microscope (7 KeV; Zeiss) using back-scatter signal detector. All image stacks obtained were then digitally processed, aligned and reconstructed in Fiji (NIH) using the TrackEM2 plugin41 (link).
The time required for the procedures were 9 hours on the first day (from the fixation to DAB development), 3–4 hours on the second day (Osmium impregnation), 5–6 hours on the third day (Dehydration), 5 hours on the fourth day (Dehydration). After 2–3 days of embedding and 4–12 hours of sectioning and staining, imaging of the samples required up to one month and tracing required 1d to a month, depending on the thickness and resolution of the sample.
The time required for the procedures were 9 hours on the first day (from the fixation to DAB development), 3–4 hours on the second day (Osmium impregnation), 5–6 hours on the third day (Dehydration), 5 hours on the fourth day (Dehydration). After 2–3 days of embedding and 4–12 hours of sectioning and staining, imaging of the samples required up to one month and tracing required 1d to a month, depending on the thickness and resolution of the sample.
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