Description
Product Usage Information
Before executing a dual staining experiment, it is suggested to optimize each primary antibody for a 1 hour primary incubation. If the recommended antigen retrieval buffer is not the same for each antibody, then it is suggested to use EDTA for both antibodies. If the primary antibodies are not compatible with the same antibody diluent, then it is advised to perform the primary incubation and detection steps sequentially. After determination of the appropriate antibody dilution, it is recommended to test the order of chromogen development. There may be differences in the quality of the results depending upon which chromogen is developed first. This is accomplished by combining the primary antibodies on two slides for the primary incubation. One slide should have DAB developed first, followed by Vibrant Red. The other slide should have Vibrant Red developed first, followed by DAB. Review the slides and determine which order of chromogens yields optimal results. Substrate Reagent Preparation: SignalStain ® DAB Substrate Kit #8059: 1. To prepare the DAB working solution, add 30 μL SignalStain ® DAB Chromogen Concentrate to 1 mL SignalStain ® DAB Diluent. Mix well before use. Working solutions are stable for up to 14 days when stored at 4ºC or up to 5 days when stored at room temperature (approximately 25ºC). While the solution may change color over that time period, it will have no effect on the quality of the staining. 2. After incubation with SignalStain ® Boost IHC Detection Reagent or other HRP-based detection systems, wash slides and incubate with the working solution at room temperature until staining develops. 1-10 minutes generally provides an acceptable staining intensity. SignalStain ® Vibrant Red Alkaline Phosphatase Substrate Kit #76713: IMPORTANT: Do not heat Vibrant Red kit components or working solution. 1. To prepare the Vibrant Red working solution, to 5 mL SignalStain ® Vibrant Red Diluent add 130 µL SignalStain ® Vibrant Red Reagent 1 and 50 µL SignalStain ® Vibrant Red Reagent 2. Mix well before use. Working solutions should be used within 15 minutes of preparation. 2. After incubation with SignalStain ® Boost IHC Detection Reagent, wash slides and incubate with the working solution at room temperature for 20-30 minutes. Staining Procedure: 1. For paraffin-embedded sections, deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series. 2. Perform antigen retrieval as recommended by the primary antibody manufacturer. If the recommended antigen retrieval buffer is not the same for each antibody, then it is suggested to use EDTA for both antibodies. 3. After washing, incubate sections in 3% H 2 O 2 diluted in water for 10 minutes. 4. Wash sections in dH2O two times for 5 minutes each. 5. Incubate sections for 30 minutes with blocking solution. 6. Remove blocking solution from sections and incubate with rabbit and mouse primary antibodies diluted in the appropriate antibody diluent as recommended by the primary antibody manufacturer. If the primary antibodies are not compatible with the same antibody diluent, then it is advised to perform the primary incubation and detection steps sequentially. 7. Wash. 8. Incubate for 30 minutes with either the AP or HRP Boost Detection Reagent previously determined to be first. 9. Wash. 10. Apply the AP or HRP-compatible substrate, matching the Boost Detection Reagent used first, and develop for the recommended time. See Substrate Reagent Preparation below. 11. Wash. 12. Incubate for 30 minutes with either the AP or HRP Boost Detection Reagent previously determined to be second. 13. Wash. 14. Apply the AP or HRP-compatible substrate, matching the Boost Detection Reagent used second, and develop for the recommended time. See Substrate Reagent Preparation below. 15. Wash. 16. Counterstain (optional), then clear and mount coverslips.
Storage
All components in this kit are stable for at least 12 months past the reference date indicated on the component label when stored at 4°C and left unused.
