石蜡切片冰冻切片免疫组化步骤

Multiple files are bound together in this PDF Package. Adobe recommends using Adobe Reader or Adobe Acrobat version 8 or later to work with documents contained within a PDF Package. By updating to the latest version, you’ll enjoy the following benefits: • Efficient, integrated PDF viewing • Easy printing • Quick searches Don’t have the latest version of Adobe Reader? Click here to download the latest version of Adobe Reader If you already have Adobe Reader 8, click a file in this PDF Package to view it. HIGH TEMPERATURE ANTIGEN UNMASKING TECHNIQUE FOR IMMUNOHISTOCHEMICAL DEMONSTRATION ON PARAFFIN SECTIONS 1. Cut and mount sections on slides coated with VECTABOND™ Reagent 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 diluted antigen unmasking solution (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 l o w e r 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. (Circle sections with the hydrophobic barrier ImmEdge™ Pen, if required.) 8. Wash sections in TBS* buffer (pH 7.4) for 1 x 5 minutes. 9. Place sections in diluted normal serum for 10 minutes. 10. Incubate sections with primary antibody. 11. Wash in TBS buffer for 2 x 5 minutes. 12. Incubate sections in an appropriate biotinylated secondary antibody. 13. Wash in TBS buffer for 2 x 5 minutes. 14. Incubate slides in ABC Elite® reagent (or RTU streptavidin/peroxidase complex). 15. Wash in TBS buffer for 2 x 5 minutes. 16. Incubate slides in DAB or other suitable peroxidase substrate. 17. Wash thoroughly in running tap water. 18. Counterstain with hematoxylin (if required), dehydrate and mount. SOLUTIONS 1. Vector® Antigen Unmasking Solution (H-3300). 2. 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. 3. 20mM TRIS/0.65mM EDTA/0.0005% TWEEN (pH9.0) Dissolve 14.4g Tris (BDH product code 271197K) and 1.44g EDTA ( S I G M A product code E-5134) to 0.55 litres of d i stilled water. Adjust pH to 9 with 1M HCI and add 0.3ml Tween 20 (SIGMA product code P-1379). Make up to 0.6 litres with distilled water. This is a 10x concentrate which should be diluted with distilled water as required (eg 150ml diluted with 1350ml of distilled water). * In most applications, 10mM phosphate, 0.15 M NaCl, pH 7.4 (PBS) can be used instead of 50 mM Tris, 0.15 M NaCl, pH 7.4 (TBS). SAFETY NOTE To ensure the correct and safe use of your pressure cooker, PLEASE READ THE MANUFACTURER’S INSTRUCTIONS. Further Suggested Products: VECTASTAIN® Elite® Mouse IgG Kit (PK-6102) DAB Peroxidase Substrate Kit (SK-4100) Vector® NovaRED™ Substrate Kit (SK-4800) Vector® Hematoxylin Counterstain (H-3401) Antigen Unmasking Solution (Citrate Salt Based) (H-3300) ImmEdge™ Pen (H-4000) VECTABOND™ Reagent (SP-1800) VectaMount™ Permanent Mounting Medium (H-5000) VECTASTAIN® ABC-AmP Chemiluminescent Kit (Western Blot Detection) (AK-6602) Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010 U.S.A. Tel: (650)697-3600 • Fax: (650)697-0339 • Email: vector@vectorlabs.com • Web site: www.vectorlabs.com V E C TO R L A B O R ATO R I E S 7/04 Protocol for immunohistochemistry - cryo sections Solutions needed:. z Fixation solution: 4 % formaline / 0.9 % NaCl / 0.5 % ZnCl2 z 0.1 M Tris-HCl, pH 7.2 z 20 % sucrose in 0.1 M Tris, pH 7.2 z 0.1 M glycine buffered with Tris-base at pH 7.4 z 0.05 M Phosphate buffer, pH 7.2 z Blocking solution: 10 % goat serum / 0.3 % Triton-X 100 / 0.05 M phosphate buffer, pH 7.2 / 0.45 M NaCl Procedure: 1. Fix tissue in fixation solution. 2. Rinse tissue in 0.1 M Tris-HCl, pH 7.2 and change buffer 3 times. 3. Incubate tissue for 12-18 h in 20 % sucrose, 0.1 M Tris, pH 7.2. 4. Freeze tissue with TISSUE-TEK using a cryostat. 5. Cut tissue (6-8 µ) and mount sections on poly-lysine coated slides. 6. Freeze the mounted sections at -20°C until use. 7. Allow frozen sections to dry at room temperature for 15 min. 8. Incubate for 30 min in 0.1 M glycine buffer. 9. Block for 1-2 h in blocking solution. 10. Incubate with primary antibody diluted in blocking solution for several hours at RT. 11. Wash with blocking solution for 30 min and change buffer several times 12. Incubate with secondary antibody diluted in blocking solution for 2-3 h at RT. Avoid bright light when working with the secondary antibody to minimize photo bleeching of the fluorescent dye. 13. Wash with blocking solution for 30 min and change buffer several times. 14. Wash with 0.05 M phosphate buffer for 30 min and change buffer several times. 15. Mount with DAKO fluorescent mounting medium. 16. Incubate for 30 min at RT. 17. Incubate over night at 4°C. 18. Incubate for 30 min at RT. 19. Seal with nail polish and microscope. Synaptic Systems GmbH | Rudolf-Wissell-Str. 28 | 37079 Göttingen | Germany Phone: +49 (0)551/505 56-0 | Internet: http://www.sysy.com | E-Mail: sales@sysy.com Protocol for immunohistochemistry - paraffin sections Deparaffinization and rehydration: 1. Incubate sections with xylene twice for 5 min. 2. Rehydrate sections through a series of ethanol concentration ranging from 100 % to 70 % (10 % steps) with a minimum of 5 min/concentration. 3. Rinse sections in PBS. Antigen retrieval: 1. Microwave sections in 10 mM citrate buffer, pH 2.5, to retrieve antigens that are masked through the embedding process. 2. Cool slides slowly to RT. 3. Rinse slides in PBS. Immunofluorescence: 1. Permeabilize sections on the slides with 0.5 % Triton X-100 (varies from antibody to antibody) in PBS for 10 min at RT. 2. Block in PBS / 2 % BSA / 0.5 % Triton X-100 for 1 h at RT. 3. Remove blocking solution and incubate sections with the primary antibody diluted in blocking solution overnight at 4°C. 4. Remove primary antibody and wash thoroughly with PBS / 0.5 % Triton X-100 3 times for 10 min. 5. Incubate with the secondary antibody for 1 h at RT. 6. Mount slices and examine microscopically. Synaptic Systems GmbH | Rudolf-Wissell-Str. 28 | 37079 Göttingen | Germany Phone: +49 (0)551/505 56-0 | Internet: http://www.sysy.com | E-Mail: sales@sysy.com