Histological Staining Techniques for Tissue Analysis

Hematoxylin-eosin (H&E) and IHC staining were performed as described previously^{38 (link)}. Briefly, for H&E staining, paraffin-embedded sections of paraformaldehyde (PFA)-fixed tissues were dewaxed and rehydrated, then processed in the order of hematoxylin staining, differentiation, bluing, dehydration, eosin staining, clearing, and addition of cover-slip. For IHC staining, the 3,3′-diaminobenzidine (DAB) staining method was used. Endogenous peroxidases were blocked using blocking solution (PV-6001, ZSGB-BIO, China). After blocking with serum, the primary antibody and biotinylated secondary antibody incubation steps, and addition of streptavidin-HRP, the DAB substrate (8059, Cell Signaling Technology, USA) was used to develop the signal color. Masson trichrome staining was performed using the kit from Solarbio (G1340, Solarbio, China) following the manufacturer’s instructions.