Super sensitive polymer hrp detection kit

Thirty cases of OSCC were retrieved from the archives of our college which included 10 cases of well differentiated (WD SCC), 10 cases of moderately differentiated (MD SCC) and 10 cases of poorly differentiated squamous cell carcinomas (PD SCC). Paraffin blocks of all cases were sectioned onto polylysin-coated slides. The avidin-biotin-peroxidase method was performed using the primary monoclonal antibodies against CD44s, standard isoform (Anti-CD44 antigen, clone –BGX- 297). Briefly, the sections were deparaffinized and washed in phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked using 0.3% solution of hydrogen peroxidase at room temperature for 5 minutes. After microwave treatment for antigen retrieval, primary antibodies were applied for 60 minutes at room temperature and washed in PBS. Linking antibody and HR-peroxidase complex (Biogenex Super Sensitive™ Polymer-HRP detection kit) were added consecutively for 20 minutes at room temperature and washed in PBS. The peroxidase activity was visualized with diaminobenzidine (DAB), applied for 5 minutes.

A microcomputed tomography (micro-CT) apparatus (SkyScan 1176; Bruker, Kontich, Belgium) was used to evaluate the calvarial defects. The micro-CT apparatus used an acceleration voltage of 80 ​kVp and 500 ​μA with a copper filter. The samples were scanned at a voxel resolution of 36 ​μm with a rotation of 0.5°. The three-dimensional images were reconstructed, and the region of interest (ROI) was analyzed using the Amira system (Thermo Fisher Scientific, USA). For histological analysis, formalin-fixed samples were trimmed and decalcified in 15% EDTA for 2–3 weeks and then were embedded in paraffin wax. Serial sections of paraffin blocks of 6-μm thickness were obtained along the coronal plane, stained with hematoxylin and eosin (H&E), and then examined under a light microscope (BX51; Olympus Corp., Japan). For immunohistochemistry detection, the sections were incubated with primary antibodies against mouse F4/80 antigen (Thermo Fisher Scientific, USA) and bone sialoprotein (Genetex, USA) at 4 ​°C overnight. After washing, the sections were coated with a super enhancer at room temperature for 20–30 ​min using the Super Sensitive™ Polymer-HRP Detection Kit (BioGenex, USA).