Methyl green solution

For histochemical analysis, poststroke brain tissue samples were fixed overnight with periodate-lysine-paraformaldehyde (PLP), cryoprotected in 30% sucrose, frozen at −80°C, and cut into 20-μm sections using a cryostat. Sections were then stained with hematoxylin and eosin (H&E), processed for immunohistochemistry, or subjected to terminal dUTP nick-end labeling (TUNEL). TUNEL was performed using an in situ apoptosis detection kit according to the manufacturer's instructions (Takara Biomedicals, Kusatsu, Japan), followed by counterstaining with methyl green solution (Wako, Osaka, Japan) as described [16 (link)].

The details of the antibodies used for the immunohistochemical analysis are listed in Supplementary Table S1. Samples were decalcified in 20% ethylenediaminetetraacetic acid for 3 weeks after micro-CT and embedded in paraffin. Sagittal paraffin sections were generated and randomly selected for hematoxylin and eosin (HE; Sakura Finetek Japan, Tokyo, Japan), toluidine blue (TB; Fujifilm Wako), and tartrate-resistant acid phosphatase (TRAP; Cosmo Bio Co., LTD., Tokyo, Japan) with methyl green solution (Fujifilm Wako) counterstaining. For immunohistochemical analysis, antibodies against iNOS, IL-1β, MMP13, SOX9, and PCNA were used with nuclear counterstaining with Mayer hematoxylin (Sakura Finetek Japan). TMJOA protocols for sample preparation are depicted in Fig. 4A, 4B.
For the immunofluorescence analysis of M2 macrophages, samples were embedded in SCEM gel (Leica, Wetzlar, Germany), and nondecalcified frozen sections were prepared according to Kawamoto’s film method.^{20 (link)} They were stained with antibodies against CD206 with anti-rabbit IgG-Alexa Fluor Plus 488 and DAPI (Sigma-Aldrich). Tissue images were captured under a universal fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). Cells were counted in at least 3 slices for each specimen.