Multiplex IHC Detection Kit (Quadruple)

1. De-paraffinizing (de-waxing) and rehydrating 1.1 Heat the slides in tissue-drying oven for 45 minutes at 60°C. Place the slides in a rack, and perform the following washes: Xylene I: 15 mins Xylene II: 15 mins 100% Ethanol: 5 mins 95% Ethanol: 5 mins 85% Ethanol: 5 mins 70% Ethanol: 5 mins 1.2 The slides are placed in a lab draught cupboard to rinse off ethanol. 1.3 Finally, wash the slides in the pure water. 2. Antigen or epitope retrieval 2.1 Add the appropriate antigen retrieval buffer (EDTA pH 9.0 or sodium citrate pH 6.0) to the microwaveable vessel. 2.2 Place the slides in the microwaveable vessel. Then place the vessel inside the microwave (1200W) . 2.3 Boil for 8 mins in microwave (1200 W) under medium heat, then stop heating for 8 mins, followed by low and medium heat for 7 mins. Other heat-induced epitope retrieval methods can also be used, e.g., heated at 120 °C 1-2 min, 100 °C 20mins or 95 °C in a water bath. Be sure to monitor for evaporation and watch out for boiling over during the procedure. Do not let the slides dry out. 2.4 After cooling down in room temperature, place the slides in PBS (pH 7.4) to wash 3 X 5 mins on a decolorizing shaker. Notes: To get best results, antigen retrieval buffer and protocol should be determined according to the tissue types and antigen types. 3. Blocking endogenous peroxidase 3.1 Add enough 3% hydrogen peroxide (H2O2) to cover the slides. 3.2 Incubate for 15 mins in the dark at room temperature. 3.3 Place the slides in PBS (pH 7.4) to wash 3 X 5 mins on a decolorizing shaker. 4. Blocking 4.1 Drain slides and then use an IHC pen to draw a circle around each sample on your slide (to hold antibody solution within the target area) . 4.2 Add 3% BSA-PBST solution (or other blocking buffer) inside the circle to cover the tissues, incubate 30 mins at room temperature. 5. Primary antibody incubation 5.1 Remove blocking buffer and add primary antibody diluted by recommended antibody diluent overnight at 4°C or 37°C for 1-2h. 6. HRP Polymer incubation 6.1 Place the slides in PBS (pH 7.4) and wash 3 X 5 mins on a decolorizing shaker. 6.2 Incubate slides with HRP Polymer (100 μl for each slice) in the dark at room temperature for 60 mins. 6.3 Wash 3 X 5 mins with PBS buffer. 7. Tyramide labeling 7.1 Apply the TSA-520 Dye Working Solution to each sample (100 μl for each slice) and incubate for 10-15 mins at room temperature. 7.2 Wash 3 X 5 mins with PBS buffer. 8. Denoise Repeat steps 2. At the end of the single stain, may add antifade mounting medium to view the slides or go on labeling another fluorescent dye. 9. Repeat Repeat steps 3-7 for another fluorescent dye (TSA-620 Dye Working Solution, TSA-690 Dye Working Solution) to each sample. 10. DAPI counterstaining 10.1 Place the slides in PBS (pH 7.4) and wash 3 X 5 mins on a decolorizing shaker. 10.2 Apply the DAPI solution (100 μl for each slice) to each sample and incubate in the dark at room temperature for 10 mins. 11. Mounting the slides 11.1 Place the slides in PBS (pH 7.4) and wash 3 X 5 mins on a decolorizing shaker. 11.2 Add antifade mounting medium (3-5 μl for each slice) to cover the section. 12. View the slides View the sample using a fluorescence microscope with appropriate filters.