Tissue faxes

Formalin-fixed tumor tissues were paraffin embedded, cut into 4-μm sections and then placed on slides. The paraffin tissue sections were incubated at 65 °C for 2 h, deparaffinized, rehydrated, and processed for antigen retrieval. Next, following by incubation with 3% H₂O₂ for 10 min to inactivate endogenous peroxidase, then the tissues were incubated overnight with the Ki67 and p-STAT3 Y705 primary antibody (1:50) at 4 °C. The next day, all tissue sections were washed three times, and then incubated with the secondary antibody followed by 3, 3′-diaminobenzidine (DAB) staining. Then the sections were counterstained with hematoxylin, dehydration, and mounted with neutral resin. The Tissue Faxes (Tissue Gnostics) and Image Pro Plus software program (Media Cybernetics, Rockville, MD) were utilized to scan all sections and calculate positive cells.

After fixation with 4% formaldehyde for at least 48 h, tumour tissues were embedded in paraffin blocks and subjected to immunohistochemistry (IHC). Serial 4 μm paraffin tissue sections were rehydrated using alcohol and TBST after being deparaffinised at 60 °C for 2 h. Next, the slides were boiled in sodium citrate buffer solution for 15 min. Afterwards, the tissue sections were treated with 3% H₂O₂ for 8 min. For IHC, tissues were hybridised with the Ki67 and p-MAPK1^T185/Y187 primary antibody (1:50) at 4 °C overnight. Then the slides were incubated with HRP-IgG secondary antibody after being washed with TBST. Finally, the slides were stained with diaminobenzidine (DAB) for 2 min and then counterstained with hematoxylin. All sections were scanned using Tissue Faxes (TissueGnostics, version 4.2) and the Image Pro Plus software program (Media Cybernetics, Rockville, MD) was used to calculate positive cells.