Lillie mayer s haematoxylin

Sections were cut from paraffin blocks of the muscle (TBD) tissue using a rotary microtome (Leica Microsystems). After sectioning, each slide was coded as a number for blind evaluation. Each number was generated using the RAND function of the Excel software, sorted in ascending order, and assigned to the slides. The tissue slides were used for staining and evaluated by an experimenter.
For HE staining, sections were cut from frozen blocks of muscle (tibialis anterior) tissue and stained with Lillie-Mayer’s haematoxylin (Muto Pure Chemicals Co., Ltd., Japan) and eosin solution (FUJIFILM Wako Pure Chemical Corporation). The inflammation score was calculated according to the criteria of Tinsley^{9 (link)}, as shown below:

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.