Envision chem kit

The immunohistochemical portion of this study, focused on expression of proteins p16 and p53, was performed on formalin-fixed, paraffin-embedded, 4-µm thick tissue sections using the avidin-biotin-peroxidase complex method. Deparaffinization of all the sections was performed through a series of xylene baths, and rehydration was performed with a series of graded alcohol solutions. To enhance immunoreactivity, microwave antigen retrieval was performed at 750 W for 30 minutes in Tris-EDTA buffer (pH 9.0). After blocking endogenous peroxidase activity with 5% hydrogen peroxidase for 10 minutes, primary antibody incubation was performed for 1 hour at room temperature. The primary antibodies were a mouse monoclonal antibodies directed against p16 (JC 8, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and p53 (DO-7, DakoCytomation, Glostrup, Denmark) used in 1:100 dilutions. An Envision Chem Kit (DakoCytomation, Carpinteria, CA, USA) was used as the secondary antibody at room temperature for 30 minutes. After washing the tissue samples in Tris-buffered saline for 10 minutes, 3,3'-diaminobenzidine was used as a chromogen, and Gill's hematoxylin counterstain was applied.

Immunohistochemical studies on p16 and Ki-67 were performed on FFPE, 4 μm thick tissue section using the avidin-biotin-peroxidase complex method. The sections were deparaffinized through a series of xylene baths, and rehydrated in a graded alcohol series. The immunoreactivity was enhanced by microwave antigen retrieval performed at 750 W for 30 minutes in Tris-EDTA buffer (pH 9.0). Endogenous peroxidase activity was blocked with 5% hydrogen peroxidase for 10 minutes and the samples were incubated with primary antibody for 1 hour at room temperature. The primary antibody was a mouse monoclonal antibody directed against p16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Ki-67 (Lab Vision, Fremont, CA, USA), used in a 1:100 dilution. The secondary antibody was used for an envision chem kit (DakoCytomation, Carpinteria, CA, USA) at room temperature for 30 minutes. The tissue samples were washed in Tris-buffered saline for 10 minutes and then stained with 3, 3′-diaminobenzidine chromogen and Gill’s hematoxylin counterstain.