Meyer s hematoxylin

The surgically resected specimen was fixed in 10% buffered formalin, processed through graded ethanol, and embedded in paraffin blocks. A 3-μm section including both normal colonic mucosa and cancerous tissue was used. Deparaffinized sections were created, and the slide was boiled for 10 min. To quench endogenous peroxidase activity, the slide was immersed in mixed methanol and H₂O₂ for 25 min at room temperature. PGE2 expression was examined using rabbit anti-human PGE2 antibody (Abcam, Cambridge, UK) and VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Laboratories, Burlingame, CA, USA). The slide was blocked by blocking serum for 20 min at room temperature and incubated with a primary antibody (rabbit anti-human PGE2 antibody; Abcam, Cambridge, UK) (1:100) overnight at 4 °C. After overnight incubation, the slide was incubated with a secondary antibody (biotinylated goat anti-rabbit antibody) for 30 min at room temperature and incubated with Reagent A and B mixed for 20 min at room temperature. It was then incubated with Histofine (Nichirei Biosciences Inc., Tokyo, Japan) for 14 min. Finally, the slide was stained with Meyer’s hematoxylin (Wako Pure Chemical Industries, Osaka, Japan) for 3 min.

Tissues were obtained from 171 patients who underwent initial surgery at Nagoya University Hospital after providing informed consent. Immunohistochemistry was performed with rabbit mAb against HS1 (CST). The histological type was specified according to the criteria of the World Health Organization classification. The slides were counterstained with Meyer's hematoxylin (Wako, Osaka, Japan). Negative controls were run on all sections in blocking buffer, generated against unrelated antigens. The intensity of immunostaining with HS1 was scored as follows: 0 (negative), 1 (weak), 2 (medium), and 3 (strong). Tumors with a final staining score of 0–1 or 2–3 were defined as having low or high expression of HS1, respectively. The scoring procedure was performed twice by two independent observers; each was blinded to the other's scores and neither had any knowledge of the clinical parameters or other prognostic factors. The interobserver concordance rate was over 95%.