Vector dab substrate

Specimens were fixed in 4% formaldehyde solution for 48 h, embedded in paraffin, and sectioned (2 µm). After deparaffinization, antigen demasking (citrate buffer, pH=6) and inactivation of endogenous peroxidases (3% H₂O₂ in methanol), permeabilization was performed (1% goat serum, 0.05% Tween-20 and 0.4% Triton-X-100 in PBS) for 10 min. After blocking for 1 h (2% goat serum in PBS), sections were incubated overnight at 4°C with primary antibodies. Following antibodies were used: anti-Cre Recombinase in 1:100 dilution (D7L7L XP^® Rabbit mAb #15036, Cell Signaling). After 1 h incubation with Horseradish peroxidase (HRP)-conjugated secondary antibody (Goat Anti-Rabbit IgG-HRP #4030-05, SouthernBiotech) in 1:200 dilution, detection was performed using Vector DAB substrate (Vector Laboratories). Afterwards, the sections were counterstained with hematoxylin.

Tissue sections were obtained from a representative paraformaldehyde-fixed paraffin-embedded tissue block of each patient's tumor. Tissue blocks containing a transmural, full-thickness section of adenocarcinoma, including the deepest pericolonic extension, were selected. Immunohistochemistry (IHC) was performed essentially as previously described [26 (link)]. Briefly, endogenous peroxidase activity of tissue sections was blocked with H₂O₂. Slides were incubated in boiling citrate buffer (pH 6.0), then maintained at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min. The sections were incubated at 4°C for overnight with anti-ARNT antibodies (diluted 1:100) (Cell Signaling Technology, Inc., Danvers, MA, USA). The reaction complexes were detected using a kit (VECTASTAIN Elite ABC kits) and visualized using a 3, 3-diaminobenzidine (DAB) substrate kit (Vector DAB substrate). Finally, the sections were lightly counterstained with hematoxylin. All IHCs were assessed by a pathologist with 10 years' experience (C-T Lee). The nuclear positivity of ARNT was counted. The IHC results of ARNT were grouped into “no expression” (no IHC reactivity or < 5% of positive cells/total tumor cells), “low expression” (faint or light brown nuclear staining in at least 5% of total tumor cells), and “high expression” (dark brown nuclear staining in at least 5% of total tumor cells).