1、AEC IMMUNOSTAINING PROCEDURERevised 01-10-111. Deparaffinize slides in old xylene I for 20 min. Discard xylene I. Pour xylene II into xylene I container. Soak 5 min.2. Pour fresh xylene into xylene II. Soak slides in fresh xylene for 5 min.3. Soak in 100% ethanol I for 5 min. Discard ethanol I. Pour
2、 ethanol II into ethanol I container.Soak 5 min. Pour fresh 100% ethanol into ethanol II container. Soak another 5 min.4. Soak in 95% ethanol for 5 min.5. Quench endogenous peroxidase in 3% H2O2 in methanol for 15 min. (20 ml hydrogen peroxide and 180 ml methanol).6. Soak in 80% ethanol for 5 min.7.
3、 Soak in 70% ethanol for 5 min.8. Soak in 50% ethanol for 5 min.9. Soak in saline for 5 min.10. Soak in warm saline (37 C) for 5 min.11. Pepsin digestion: incubate slides in 0.4% pepsin in 0.01 N HCl for 15 min. at 37 C. (0.8 gm pepsin, 0.333 ml 6 N HCl, 200 ml saline, prewarmed to 37 C for 30 min).
4、12. Soak in 0.75% BRIJ-PBS for 5 min. twice (2.5 ml 30% BRIJ 35 per liter PBS). 13. Block nonspecific binding with 0.5% Na caseinate in BRIJ-PBS for 10 min. in the slide moat.14. Drain and without rinsing apply the properly diluted primary antibody to the tissues for 30min. at 37 C.15. Soak slides i
5、n BRIJ-PBS for 5 min. twice.16. Treat tissues with Dako EnVision Rabbit peroxidase (catalog # K4003) solution at 37 Cfor 30 min.17. Soak slides in BRIJ-PBS for 5 min. twice.18. Apply Dako AEC substrate chromogen (catalog # K3469) solution to slides at 37 C for 15 min.19. Collect chromogen in hazard
6、waste bottle, and soak slides in BRIJ-PBS for 5 min.20. Rinse slides in running tap water for 2 min. Rinse in de-ionized water for 2 min. twice.21. Counterstain with Mayers Hematoxylin for 1 min.22. Wash slides in running tap water for 1 min.23. Soak slides in Scotts tap water substitute (MgSO4.7H2O 20 gm, NaHCO3 2 gm, water 1liter) for 1 min.24. Soak in running tap water for 1 min. Soak in de-ionized water for 2 min. twice.25. Apply Crystal Mount and incubate slides overnight at room temperature.26. Apply Permount and cover slip, if desired. Staining.wpd
