Super sensitive non biotin polymer hrp detection system

Immunohistochemical staining of tumor tissue samples from patients with adenocarcinoma was conducted as previously described. Briefly, sections for analysis of Slug or MDA-9/Syntenin protein expressions were first autoclaved in Trilogy Solution (Cell Marque Corp) or Antigen Retrieval Citra Solution (Biogenex, San Ramon, CA) at 121°C for 10 min. The samples were subsequently treated with 3% H₂O₂-methanol and incubated with DakoCytomation Dual Endogenous Enzyme Block (DakoCytomation) for 10 min, Ultra V Block (Lab Vision Corporation) for 10 min, antibody-dilution buffer (Ventana Medical Systems, Inc., Tucson, AZ) for 10 min, and with anti-Slug (1:75, ABGENT) antibody at room temperature or the anti-MDA-9/Syntenin antibody (1:100, Santa Cruz Biotechnology) overnight at 4°C.
Immunostaining was detected using a Super Sensitive Non-Biotin Polymer HRP Detection System (BioGenex, San Ramon, CA) according to the manufacturer's protocol.

The sections used for IHC analysis of ID4, SLUG, E-cadherin, and Ki67 protein expression were first autoclaved in Antigen Retrieval Citra Solution (BioGenex, Milmont Dr, Fermont, CA, USA) at 121 °C for 10 min. The samples were then treated with 3% H₂O₂; and sequentially, subjected to incubation with 0.1% BSA for 1 hour, and then with a monoclonal anti-ID4 antibody (Santa Cruz Biotechnology, Dallas, TX, USA; 1:100), a polyclonal anti-SLUG antibody (Santa Cruz Biotechnology; 1:100), a monoclonal anti- E-cadherin (BD; 1:100), and a rabbit polyclonal anti-Ki67 (Abcam, Cambridge, UK; 1:500) overnight at 4 °C. Detection of the immunostaining was performed using the Super Sensitive Non-Biotin Polymer HRP Detection System (BioGenex), according to the manufacturer’s instructions.

For immunohistochemistry [14 (link)], paraffin sections were treated with hydrogen peroxide to inactivate endogenous peroxidases. Antigen retrieval was performed in a microwave in 10 mM citrate buffer at pH 6.0. Sections were fixed with paraformaldehyde followed by permeabilization and blocking. Sections were then incubated in anti-URG11 (Santa Cruz) antibody overnight at 4°C, and a secondary antibody was used to detect protein expression. Immunostaining was analysed with the Super Sensitive Non-Biotin Polymer HRP Detection System according to the manufacturer's instructions (BioGenex, San Ramon, Canada).
For analysis, randomly selected five fields of the staining were captured; the grade values were determined by the percentage of positive stained cells and the strength of URG11 staining [15 (link)]. Briefly, the ratio of positive cells scored 0 for staining of ≤1%, 1 for staining of 2 to 25%, 2 for staining of 26 to 50%, 3 for staining of 51 to 75%, and 4 for staining >75%. Staining intensity was graded as follows: negative: 0; weak signal: 1; middle: 2; and strong: 3. Then the total score resulting from the multiple value of positive percentage score and strength score is as follows: 0-1: grades negative (-); 2-4: weak positive (+); 5-8: middle (++); and 9-12: strong (+++).