Histological Sample Preparation for ASEM

Hind limbs were fixed with 4% PFA in 0.1 M CB (pH 7.4) at 4 °C for 1 day and further fixed with 1% glutaraldehyde (GA; Nisshin EM, Tokyo, Japan) in 0.1 M CB at 24 °C for 15 min. The samples were washed several times with double-distilled water (DDW), embedded in 4% agar, and cut with a PRO7 linear slicer (Dosaka, Kyoto, Japan) to obtain 200-μm-thick sections or a single cut tissue. The tissues were stained with aqueous 0.067% haematoxylin solution (Lillie-Mayer’s Haematoxylin, Muto Pure Chemicals, Tokyo, Japan) for 1 min at 24 °C and immediately washed with DDW. The sections were then stained with an aqueous solution of 0.05% eosin (diluted from 1% eosin alcohol solution, Muto Pure Chemicals) for 1 min at 24 °C and washed several times with DDW. Samples were then placed on an SiN-windowed specimen dish and immersed in the observation buffer required for ASEM imaging. An Olympus SZX12 stereomicroscope was used to facilitate these operations. Tissue sections were counterstained with 2% phosphotungstic acid (PTA, TAAB Laboratories Equipment Ltd., Aldermaston, UK) at 24°C^{35 (link)} for ASEM.