2.12. Histopathological assay

Tumor‐bearing mice were euthanized with isoflurane (MSD Animal Health). The tumor masses were collected. Tumor masses were fixed at 4°C for 24 h in 4% paraformaldehyde (163–20145) (Fujifilm Wako Pure Chemical Corp.), were embedded in paraffin blocks, and were sectioned at 5 µm thickness using a microtome to prepare tissue slides. After deparaffinized and rehydration, tissue slides were stained with hematoxylin and eosin (H&E) or were immunostained. For immunostaining, the slides were heated for 10 min at 95 to 100°C in 10 mM sodium citrate buffer (pH 6.0) to recover antigenicity and blocked with blocking solution consisting of 0.05% (v/v) Tween 20 (P1379) (Sigma‐Aldrich Corp.) in phosphate buffered saline (PBS‐T) supplemented with 2% (w/v) bovine serum albumin (A5611) (Sigma‐Aldrich Corp.) for 1 h at 20°C. Thereafter, sections were reacted with an anti‐Ki‐67 antibody (LS‐B6433) (LifeSpan BioSciences Inc.) at a dilution of 1:200 in blocking solution for 24 h r at 4°C. After washing with PBS‐T, the sections were reacted with an anti‐Mouse IgG (H + L), HRP Conjugate (W4021) (Promega Corp.) at a dilution of 1:2000 in blocking solution for 1 h at 20°C. After washing with PBS‐T, Ki‐67‐positive nuclei were visualized with DAB chromogen substrate (K3468) (Agilent Technologies Inc.). To detect expression of green fluorescent protein (GFP) in tumor tissue, the collected tumor masses were fixed in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corp.), embedded in OCT compound (4583) (Sakura Finetechnical Co. Ltd.), and frozen at −80°C. After cryosections were prepared from the frozen blocks at 20 µm thickness using a cryostat (Leica CM1950; Leica Biosystems), nuclei were counterstained with mounting medium with DAPI (4′,6‐diamidino‐2‐phenylindole) (H‐1200) (Vectashield; Vector Laboratories, Burlingame, CA) and were observed using BZ‐X fluorescence microscopy (Keyence Co.). Paraffin‐embedded lung sections from two lung cancer patients, diagnosed as squamous cell carcinoma or adenocarcinoma, were kindly provided by Tohoku University Hospital (Sendai, Japan). The specimens were sectioned at 5 µm thickness and were stained with H&E or were immunostained as described above. Briefly, for detection of STC‐1, sections were reacted with an anti‐Stanniocalcin‐1 Rabbit‐Poly (GTX129092) (Genetex Inc.) at dilution of 1:200 in blocking solution for 24 h at 4°C. After washing with PBS‐T, the sections were reacted with a goat anti‐rabbit Ig FITC conjugated (NB7182) (Bio‐Techne) at a dilution of 1:2000 in blocking solution for 1 h at 20°C. After immunofluorescent staining, nuclei were counterstained with Vectashield mounting medium with DAPI (Vector Laboratories) and were observed using BZ‐X fluorescence microscopy (Keyence Co.). This study was approved by the Institutional Review Board of Tohoku University Graduate School of Medicine (Sendai, Japan).