After anesthetization, two adult specimens were fixed in Bouin’s solution overnight. The subsequent preparation followed the protocol by Sombke et al. [33 (link)]. Preparations were rinsed in several changes of PBS (phosphate buffered saline, 0.1 M, pH 7.4), dehydrated in a graded ethanol series and incubated in a 1% iodine solution (iodine resublimated in 99% ethanol; Carl Roth #X864.1) for 12 h. Preparations were rinsed several times in pure ethanol and critical-point-dried. Finally, samples were fixed on insect pins with super glue. Scans were performed with a Zeiss Xradia MicroXCT-200. Dissected walking legs (pair 10) were scanned with a 10× objective lens resulting in a 2.19 μm pixel size; ultimate legs were scanned with a 4× objective lens unit resulting in 5.05 μm pixel size. Dissected ventral nerve cord ganglia associated with walking legs were scanned with a 20x objective lens resulting in a 0.93 μm pixel size (compare also Schendel et al. [24 ]). The ultimate leg associated ganglion 15 was scanned using a 20× objective lens resulting in a 1.09 μm pixel size. Tomography projections were reconstructed using the XMReconstructor software (Zeiss Microscopy) resulting in image stacks (TIFF format). All scans were performed using binning 2 (resulting in noise reduction) and subsequently reconstructed using binning 1 (full resolution) to avoid information loss.
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