Pas reagent

The lung tissues were fixed with 4% paraformaldehyde at 4 ˚C overnight, washed with PBS and processed into paraffin blocks. Tissues sections were stained with periodic acid-Schiff (PAS) reagent (Beijing Solarbio Science & Technology Co, Beijing, China) for analyzing the content of glycogen. Positive glycogen staining was visualized in red or purple color. The sections were observed under a light microscope and the images were analyzed by Image-Pro Plus III (Media Cybernetics, Inc., Rockville, MD, USA) to obtain the mean gray value.

Liver tissue was embedded in paraffin. Liver sections (4 μm) were stained with PAS reagent (Solarbio, Beijing, China) to assess glycogen. Slices were analyzed under light microscopy, and images were captured with a digital camera.
Additionally, SHED engraftment in liver tissue was detected by immunohistochemistry in prepared sections as follows. Briefly, the sections were deparaffinized, rehydrated and submitted to Tris–EDTA, pH 9.0 antigen retrieval solution (Zhongshan Golden Bridge Biotechnology, Beijing, China) in a microwave for 30 min. Next, 3% hydrogen peroxide (Zhongshan Golden Bridge Biotechnology, Beijing, China) was used to block the endogenous peroxide. Then, the sections were incubated with human DNA PKcs primary antibody (Proteintech, Chicago, IL, USA) at 4 °C overnight. After a thorough rinse with PBS, sections were incubated with a secondary antibody using a horseradish peroxidase polymer system (Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature for 30 min. The detection step was performed with diaminobenzidine (Zhongshan Golden Bridge Biotechnology, Beijing, China). Finally, sections were counterstained with hematoxylin to distinguish cell nuclei.