Envision system peroxidase kit

Sections (5 µm) of formalin-fixed, paraffin-embedded ISCC specimens were mounted on poly-L-lysine (Sigma, St. Louis, MO, USA) coated slides and processed for conventional histological assessment by H&E staining and Immunohistochemistry (IHC) after de-waxing and then dehydrating. Antigen retrieval treatment was done in citrate buffer in microwave and then cooled for 20–30 min. After washing in 0.01M Tris-buffered saline (pH 7.4), sections were incubated with 1% BSA followed by overnight incubation at 4 C with primary polyclonal anti-human antibody of Notch-1 and Notch-3 (ab 8925, ab23426 Abcam) at a dilution of 1∶200 and 1∶500 respectively. An EnVision+System peroxidase kit (DAKO, Carpinteria, CA) was used for staining. Sections were then counterstained with Mayer's haematoxylin solution, dehydrated in ascending concentrations of ethanol (30% to 100%), cleared in xylene and mounted with DPX. For each target staining, all slides were processed at the same time. The stained tissue slides were examined under an Olympus microscope (model BX-51, Olympus America, Inc., Melville, NY) with ‘‘Olysia Bio-report’’ software linked with a personal computer. The primary antibody was replaced with isotype-specific IgG as a negative control.

Fine sectioning (5 μm) was done in formalin-fixed paraffin-embedded specimens on poly-L-lysine (Sigma, St. Louis, MO, USA) coated slides, followed by immunohistochemistry (IHC). The antigen retrieval for Cyclin D1 was performed in 10 mM tris-EDTA buffer (pH-9.0) at 900W for 15 min/360W for 5 min. The sections were consecutively washed in Tris buffer saline (TBS; 0.1M; pH: 7.4) and then incubated with primary antibody of Primary rabbit monoclonal of Cyclin D1 (monoclonal, M3635, Dako cytomation Glostrup, Denmark; 1:200 dilution) overnight followed by the use of polymer based secondary antibody (Envision System peroxidase kit, DAKO, Carpinteria, CA) for 1 hr at room temperature after washing with TBS. Previous studies from our laboratory have mentioned the complete methodological details^{9 (link),10 (link)}.

Mouse anti‐YWHAZ monoclonal antibodies (NBP1‐61188) were utilized for IHC studies using standard protocols with an Ab dilution of 1:100. Chromogen color was developed using an EnVision+System peroxidase kit (DAKO, Carpinteria, CA, USA) and the immunointensity of YWHAZ staining was scored independently by two pathologists and labeled as negative (0), weakly positive (+1), moderately positive (+2), strongly positive (+3), or intensely positive (+4). In cases where there was not consensus, a third investigator was invited to score the sample, and the final score was determined by the majority scores.