Mounting solution

The liver tissues were fixed overnight in 4% PFA, embedded in paraffin or O.C.T compound (Leica) and sectioned sequentially at 8–15 μm. The sections were permeabilized with 0.2% Triton X–100 and blocked with CAS–Block solution (Invitrogen) to prevent non–specific binding. For immunofluorescence staining analysis, the sections were incubated with primary antibodies diluted in CAS–Block solution overnight at 4°C. After the primary antibody incubation, the sections were washed with DPBST three times for 10 min each. The following secondary antibodies were diluted in PBS and applied for 60 min. Images were visualized by fluorescence microscopy (Leica). The primary antibodies used for analysis are listed in S3 Table. The nucleus was stained with hematoxylin (Sigma) and blued in 0.2% ammonium hydroxide. Tissue was then placed in Eosin Y solution (Sigma) and followed by several washings and dehydrating. Slides were mounted with a mounting solution (Leica).

For histological analysis, the rats were deeply anesthetized and perfused with PBS followed by 4% paraformaldehyde as well. The spinal cords were isolated and post-fixed in 4% paraformaldehyde overnight, and then immersed in 30% sucrose for 3 days. The tissues were embedded in frozen section compound (Leica) and sectioned at 16 μm in sagittal plane by a cryostat. IHC was performed as previously described (Kim et al., 2015 (link)). The primary antibodies used for IHC are listed in Supplementary file 1.
For hematoxylin and eosin (H and E) staining, the spinal cords were embedded in paraffin blocks. The paraffin blocks were sectioned at 4 μm in sagittal plane. Sections were immersed in Harris hematoxylin solution (Sigma) for 2 min to stain nucleus. Slides were then immersed briefly in 1% acid alcohol (1% HCl in 70% ethanol) and blued in 0.2% ammonium hydroxide, followed by staining with eosin Y solution (Sigma) for 30 s. Each step was followed by several washings with distilled water. The slides were dehydrated with ethanol, cleared with xylene, and mounted with mounting solution (Leica).