高温处理＋胰酶消化修复抗原

高温处理＋胰酶消化修复抗原 High Temperature Antigen Unmasking Technique followed by Trypsin Digestion 1. Cut and mount sections on slides coated with a suitable tissue adhesive. 2. Deparaffinise sections and rehydrate to distilled water. 3. Place sections in 0.5% hydrogen peroxide/methanol for 10 minutes (or use other appropriate endogenous peroxidase blocking procedure). Wash sections in tap water. 4. Heat 1500ml of the recommended unmasking solution (0.01M citrate buffer, pH6.0 unless otherwise indicated overleaf) until boiling in a stainless steel pressure cooker. Cover but do not lock lid. 5. Position slides into metal staining racks (do not place slides close together as uneven staining may occur) and lower into pressure cooker ensuring slides are completely immersed in unmasking solution. Lock lid. 6. When the pressure cooker reaches operating temperature and pressure (after about 5 minutes) start a timer for 1 minute (unless otherwise indicated on the data sheet). 7. When the timer rings, remove pressure cooker from heat source and run under cold water with lid on. DO NOT OPEN LID UNTIL THE INDICATORS SHOW THAT PRESSURE HAS BEEN RELEASED. Open lid, remove slides and place immediately into a bath of tap water. 8. Place slides in distilled water to preheat the sections to 37oC for a minimum of 5 minutes. 9. Incubate sections in trypsin solution at 37oC for 30 seconds. 10. Rinse sections in running tap water. 11. Proceed with immunohistochemistry protocol. 12. Wash sections in TBS* buffer (pH 7.6) for 1 x 5 minutes. 13. Place sections in diluted normal serum (or RTU Normal Horse Serum) for 10 minutes. 14. Incubate sections with primary antibody. 15. Wash in TBS buffer for 2 x 5 minutes. 16. Incubate sections in an appropriate biotinylated secondary antibody. 17. Wash in TBS buffer for 2 x 5 minutes. 18. Incubate slides in ABC reagent (or RTU streptavidin/peroxidase complex). 19. Wash in TBS buffer for 2 x 5 minutes. 20. Incubate slides in DAB or other suitable peroxidase substrate. 21. Wash thoroughly in running tap water. 22. Counterstain with haematoxylin (if required), dehydrate and mount. SOLUTIONS 1. TRYPSIN SOLUTION 1. Preheat the following to 37oC using a water bath: (i) 200ml of TBS (ii) 200ml of distilled water. 2. Dissolve 0.2g Trypsin 250 and 0.2g Calcium Chloride in the 200ml of TBS. 3. Once the trypsin solution is at 37oC, pH to 7.8 with 1M sodium hydroxide. 2. 0.01 M CITRATE BUFFER (pH 6.0) Add 3.84 grams of Citric acid (anhydrous) to 1.8 litres of distilled water. Adjust to pH 6.0 using concentrated NaOH. Make up to 2 litres with distilled water. 3. 1mM EDTA (pH 8.0) Add 0.37g of EDTA (SIGMA product code E-5134) to 1 litre of distilled water. Adjust pH to 8.0 using 1.0M NaOH. * In most applications, 10mM phosphate, 0.15 M NaCl, pH 7.6 (PBS) can be used instead of 50 mM Tris, 0.15 M NaCl, pH 7.6 (TBS). SAFETY NOTE To ensure the correct and safe use of your pressure cooker, PLEASE READ THE MANUFACTURER’S INSTRUCTIONS.