protozoan staining protocol.doc

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 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. 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).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 30 min. at 37 C.15. Soak slides in BRIJ-PBS for 5 min. twice.16. Treat tissues with Dako EnVision Rabbit peroxidase (catalog # K4003) solution at 37 C for 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 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 1 liter) 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 inc