Prestaining Steps: Cut 4-5 micron sections on a chrome alum-gelatin filled water bath and pick up on Fisherbrand Superfrost*Plus (Cat #: 12-550-15) slides. Using tonsil tissue as a kit positive control Dry overnight 3 days at 37 C and 65 for 30 minutes (in the microwave for 2 minutes 30 seconds on Power Level 7, if necessary) List tests on the Daily Worksheet. Check antibody information chart to ensure correct time, temperature and enzymes/antigen retrieval are used. Dewax and Rehydrate: Deparaffinize by immediately immersing warm slides into xylene. Xylene 3 changes for 5 minutes each Absolute Ethanol 3 changes for 5 minutes each 90% Ethanol 1 change for 1 minute 70% Ethanol 1 change for 1 minute Page 1 of 6
Proteolytic Digestion and Target Retrieval: Digestion of Pepsin: Incubate slides with 0.5 % Pepsin 20 min Wash in cold running water for 3 min Bath in TBST buffer for 5 min Digestion of Proteinase K: Incubate slides with 10 mg/ml proteinase K for 10 min Wash and bath in ddh 2 O for 5 min Target antigen retrieval: Place 50 ml Retrieval Solution (Dako, Cat#: S1700) in Coplin jar Add distilled water to the pressure cooker (Dako, Cat# CD0300) to depth at least 5 cm Place the gasket into position around the rim of the bowl and Place the red weight microwave on the vent hole Position the cooker at centre of the microwave oven Heat the water using full power until the yellow pressure indicator valve rises (taking about 15 17 min) Turn off the microwave oven and allow the yellow indicator to fall Remove the pressure cooker from the microwave oven (using thermal protective gloves) Carefully open the pressure cooker and place the Coplin jar in the centre of cooker Page 2 of 6
Seal the cooker and re-cook on full power until the yellow pressure indicator rises and a constant hissing sound is heard (may taking 2 3 min) At this point, the timing should start: o For Chlamydia pneumoniae (Dako RR402) 5 min When heating is completed, allow the pressure cooker to cool naturally, by leaving it in the microwave over the yellow indicator has fallen Open the cooker and cool down for 20 min Remove the slides from the retrieval buffer and place them IMMEDIATELY into TBST buffer (DO NOT LET THE HEATED SECTIONS DRY OUT) Page 3 of 6
Staining Procedure: Bath slides in TBST and wash well in distilled water for 5 min Using Kimwipe carefully wipe around specimen to remove the water to facilitate the use of the PAP pen. Draw a complete circle around the sections with the PAP pen to confine the subsequent liquid and save the reagents. Keep the section as wet as possible. Place slides in moisture chambers Avidin-Biotin Block System (Dako, Cat# X0590) Optional For antigen retrieval slides normally apply this step Avidin Block for 10 min and rinse and bath with ddh 2 O for 5 min Biotin Block for 10 min and rinse and bath with ddh 2 O for 5 min H 2 O 2 block for 5 min (Bottle 1) Rinse ddh 2 O and bath in TBST twice for 5 min Protein block for 5 min (Bottle 2) DO NOT RINSE Primary antibody, positive or negative antibodies for 15 30 min (Concentration and duration depending on individual antibody) After adding primary reagent, prepared Streptavidin Biotin complex Link antibody for 15 min o Mouse -- working dilution (Bottle 4) o Rabbit 1:1000 (Dako, Cat#: E0353) Streptavidin biotin complex for 15 min Page 4 of 6
Amplification reagent for 15 min (Bottle 8) Streptavidin peroxidase for 15 min (Bottle 9) Toward the end of this step, a fresh batch of DAB was prepared Bath in TBST once for 5 min and bath distilled water for 5 min DAB for 1-5 min depending on individual antibody Bath distilled water for 5 min Counterstain Hematoxylin (Dako, Cat#: S3309) for 5 min Blue in 0.08% NH4OH for 1 min Wash in warm tap water for 5 min Dehydrate, Clear, and Mount 3 Changes of 100% EtOH, 20 dips each 3 Changes of xylene, 20 dips each Mount and coverslipped with nonaqueous permanent mounting medium (Fisher, Cat#:SP15-100) Page 5 of 6
REAGENT PREPARATION: Wash Buffer Solution Tris Buffer Saline-Tween20 (TBST): -0.05 M Tris-HCl ph 7.6, containing 0.3 M NaCl and 0.1% Tween 20 1 L 2L 1M Tris-HCl stocks at ph 7.6 50ml 100ml NaCl 17.5 g 35.5 g Tween 20 1 ml 2 ml Water upto 1L upto 2L Store at 2 8 C 0.5 % Pepsin: 0.5 g pepsin 100 ml 0.01N HCL 0.08 % NH4OH 2.9 ml of 28% NH4OH make up to 1000 ml Citrate Buffer: (or purchase a 500 ml of ready to-use DAKO Target Retrieval Solution (Cat#: S1700). The Solution can be used twice and stored at 4 C after the first used) Stock Solution 1= 0.1M citric acid (C 6 H 8 O 7 H 2 O) 21.01g citric acid dissolve in 1000ml ddh 2 O Stock Solution 2= 0.1M Sodium citrate 29.41G Trisodium citrate dihydrate (Na 3 C 6 H 5 O 2H 2 O) dissolve in 1000ml dh 2 O 0.01m citrate Buffer (ph 6.0) working solution: Mix 9ml of Solution 1 and 41 ml of Solution 2 Add 450 ml of ddh 2 O ph should be 6.0 (±0.1) Page 6 of 6