Avidin Biotin Complex (ABC) Method

1. Paraffin section or frozen section to water and rinse in PBS-Tween 20 for 2x2 min.
2. Antigen Retrieval: perform antigen retrieval if necessary.
3. Serum Blocking: incubate sections in normal serum – species same as secondary antibody. Note: since this protocol uses avidin-biotin detection system, avidin/biotin block may be needed based on tissue type.
4. Primary Antibody: incubate sections in primary antibody at appropriate dilution in antibody diluent (Cat# IW-1000 or IW-1001) for 1 hour at room temperature or overnight. Note: No serum blocking is needed if antibody diluent is used.
5. Rinse in PBS-Tween 20 for 3x2 min.
6. Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes at room temperature. Note: For acetone fixed frozen sections, perform this peroxidase blocking step using 0.3% H2O2 in methanol prior to primary antibody incubation to avoid tissue destruction.
7. Rinse in PBS-Tween 20 for 3x2 min.
8. Secondary Antibody: incubate sections in biotinylated secondary antibody in PBS for 30 minutes at room temperature.
9. Rinse in PBS-Tween 20 for 3x2 min.
10. Detection: incubate sections in ABC-peroxidase solution for 30 minutes at room temperature.
11. Rinse in PBS-Tween 20 for 3x2 min.
12. Chromagen/Substrate: incubate sections in peroxidase substrate solution.
13. Rinse in PBS-Tween 20 for 3x2 min.
14. Counterstain with counterstain solution if desired.
15. Rinse in running tap water for 2-5 minutes.
16. Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2x3min.
17. Clear in xylene for 2x5min.
18. Coverslip with mounting medium.