Universal detection kit

Immunostaining was done using the standard avidin-biotin peroxidase method. Paraffin sections were de-paraffinized in xylene, and then rehydrated using descending grades of ethanol. Afterwards, antigen retrieval was done by treating the sections with 0.01 M citrate buffer (pH 6.0) for 30 minutes. After rapid rinsing in phosphate buffered saline (PBS), the sections were incubated in 0.3% hydrogen peroxide for 30 minutes to stop endogenous peroxidase enzyme (Dako ko411 kit). The sections were then treated with 5% horse serum for 2 hours at room temperature to inhibit the non-specific immunoreactions.
Primary monoclonal antibodies were incubated overnight in a humidity chamber (Table 1). After washing the sections in PBS, they were then incubated with biotinylated secondary antibodies for 30 minutes, and then treated with avidin-biotin peroxidase complex for another 30 minutes, according to the manufacturer’s instructions (Universal Detection Kit, Dako, Denmark). Finally, after visualization of the immune reaction using 3,3 - diaminobenzidine tetra hydrochloride (DAB, Dako K0114 Kit) for 5 minutes, the slides were counterstained with Mayer’s hematoxylin and mounted.
All the steps were performed at room temperature. Negative controls for all markers by omission of the primary antibody were performed. The surrounding normal salivary tissue was used as internal control.