Avidin biotin horseradish peroxidase complex abc kit

The expression of p53 protein and CD8 + cells in tumor tissue sections from different in vivo treatment groups were assessed by immunohistochemistry. Tumor sections were fixed in 4% buffered formaldehyde solution and embedded in paraffin. Paraffin-embedded sections were deparaffinized, rehydrated, and washed in distilled water. In order to retrieve the antigen, tumor tissue sections were incubated in 10 mM citrate buffer (pH = 6) for 30 min, washed in PBS, and immersed in 0.3% hydrogen peroxide (H₂O₂) for 20 min, then incubated in blocking buffer (5% normal goat serum and 1% BSA) for 60 min. Tissue sections were then incubated with the appropriate primary antibodies (PBS solution supplemented with 0.3% Triton X-100) at 4 °C overnight in a humid chamber. After being rinsed with PBS, the samples were incubated with biotinylated secondary antibody at room temperature for 30 min, rinsed again with PBS, and incubated with the avidin-biotin-horseradish peroxidase complex (ABC kit, Vector Laboratories, Inc). After being washed again, stains were processed with the diaminobenzidine peroxidase substrate kit (Impact DAB, Vector Laboratories, Inc) for 3 min. Sections were evaluated using a Leica Microsystem after being counterstained with hematoxylin (Sigma), dehydrated, and mounted.

Bovine serum albumin, diethylnitrosamine, ethanol, and DMSO were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MA, USA). Rabbit polyclonal GST-placental form (GST-P) antibody was obtained from MBL (Nagoya, Japan). Mouse monoclonal proliferating cell nuclear antigen (PCNA) antibody was obtained from BioLegend (Santiago, CA, USA). EnVision Doublestain system was obtained from Dako (Glostrup, Denmark). Avidin-biotin-horseradish peroxidase complex (ABC) kit was obtained from Vector Laboratories (Burlingame, CA, USA). Apoptosis Detection Kit was obtained from Merck (Cambridge, MA, USA)). Any other chemicals were of analytical grade and were used without any purification.