Mayers hematoxylin

Tissue was also fixed in 10% formaldehyde. According to the standard protocol, the paraffin sections were deparaffinized. After repairing antigen and blocking endogenous peroxidase act, tissue sections were blocked in 3% BSA. Tissue sections were incubated with primary antibodies (Ki67, cleaved caspase3, cleaved PARP1, Servicebio, Wuhan, China) at 4 °C overnight, followed by conjugated secondary antibodies (Servicebio, Wuhan, China) and diaminobenzidine (DAB, Servicebio, Wuhan, China). Then nuclei counterstaining was performed with Mayers hematoxylin (Servicebio, Wuhan, China). Finally, the tissue sections were dehydrated and sealed.

Tissue samples fixed in 4% paraformaldehyde solution that buffered by 0.1 M phosphate solution for 24 h and then decalcified in 4% EDTA for 4 weeks. Tissue samples were stained by Mayer’s hematoxylin (Servicebio, China) and eosin (Servicebio, China), Goldner, Sirius Red, MSB (Pythonbio, China), CD68 antibody (1:100; Abcam), inducible nitric oxide synthase (iNOS) antibody (1:50; Abcam), α-SMA antibody (1:100; Affbiotech), LC3 antibody (1:100; Cell Signaling Technology), and goat anti-mouse/rabbit secondary antibody (GeneTech, China) for different purposes. Images were captured using Aperio AT2 system (Leica Aperio AT2, Germany) and semiquantification was conducted using ImageJ software (1.46) and ImageScope software.