Multiplex Fluorescent (5 colors) IHC Staining Kit

Cat. No.: IHCT003
Detection method: Fluorescent
Section type: FFPE
Size: 100T
Storage and Stability: Store protected from light at 2–8℃ for 12 months.

Product Components

Principle

Tyramide Signal Amplification (TSA) is an enzymatic detection method that utilizes horseradish peroxidase (HRP) to achieve high-density in situ labeling of target proteins. Its principle involves the peroxidase-driven reaction of tyramide: under the catalysis of HRP and hydrogen peroxide (H2O2), the tyramide-fluorophore conjugate is activated. The activated fluorescent substrate covalently binds to protein residues (specifically tyrosine residues) near the target protein, resulting in massive deposition of fluorophores at the antigen-antibody binding site and thereby amplifying the signal. Through antigen retrieval, antibodies from the previous round (non-covalently bound) are removed, while the fluorophores remain stably attached to the protein. Subsequent rounds of staining proceed sequentially. After all antibody incubations are completed, nuclear staining is performed, followed by mounting and imaging. Since only a single antibody is incubated in each round, there is no need to worry about antibody cross-reactivity or species matching between primary and secondary antibodies, thereby eliminating the traditional limitations of antibody species sources in immunofluorescence experiments. The Retrieval/Stripping 2-in-1 Buffer in this kit outperforms conventional retrieval solutions by significantly preventing "signal bleeding" and "over-retrieval" artifacts. The TSA Buffer for Rapid Reaction removes dependence on H2O2, shortening reaction time from 5–20 minutes to 10 seconds~10 minutes. The Rapid Mounting Medium incorporates bubble-inhibiting and enhanced antifading formulations, effectively preventing bubble formation during mounting and enabling TSA fluorescence signals to be preserved for 6 months~2 years. This kit provides all necessary reagents in a single package, significantly reducing operation time and simplifying complex procedures.

Protocol Summary

1. Deparaffinization And Rehydration: Immerse slides in fresh xylene for 10 minutes and then repeat two more times using separate containers. Immerse slides sequentially in 100%, 100%, 95%, 95%, 85%, and 75% ethanol solutions for 5 minutes each. Rinse slides with ddH2O for 5 minutes.
   
2. Antigen Retrieval: Submerging slides completely in retrieval buffer. Perform retrieval via microwave or pressure cooking. Preparation of 5L Retrieval/Stripping 2-in-1 Buffer Working Solution: Add 16g powder to 92ml ddH2O and stir until fully dissolved. Dilute the concentrate with ddH2O to a final volume of 5L. Set microwave to high power and heat until the buffer reaches boiling point, hold for 20 seconds. Immediately switch to low power and maintain gentle boiling for 5 minutes. Turn off microwave and allow slides to cool naturally to room temperature.
3. Rinse 3 times with PBS Buffer for 5 minutes each.
4. Block Endogenous Peroxidase: Drain the liquid off the slides and then use a hydrophobic IHC pen to draw circles on the slides around tissue sections. Add 3% Hydrogen Peroxide directly on slides, covering the whole tissue and incubate for 10 minutes at RT.
5. Rinse 3 times with PBS Buffer for 5 minutes each.
6. Blocking: Drain the liquid off the slides and then block with Blocking Buffer for 30 minutes at RT.
7. Primary Antibody Incubation: Drain the liquid off the slides and then incubate slides with primary antibody working solution diluted to an appropriate concentration, overnight at 4℃ or 1 hour at 37℃.
8. Rinse 3 times with PBS Buffer for 5 minutes each.
9. Secondary Antibody Incubation: Drain the liquid off the slides and then incubate slides with HRP labeled secondary antibody for 30 minutes at RT.
10. Rinse 3 times with PBS Buffer for 5 minutes each.
11. Chromogenic Reaction and Signal Amplification: Dilute the TSA dye in TSA Buffer for Rapid Reaction to prepare the working solution (refer to the Fluorophore Spectral Data for specific dilution ratios). Apply 50-200 μL of working solution to completely cover the tissue section. Incubate at RT for 60 seconds. Terminate the reaction by immersing slides in PBS for 2 minutes. Note: It is recommended to perform microscopic examination after each staining round to assess the results. If background fluorescence is high, immerse the slide in PBS for a period, then re-examine. If signal intensity is insufficient, reapply the diluted working solution (1:20-1:50) and extend the reaction time appropriately. Optimal reaction time: Approximately 60 seconds, no more than 20 minutes.
12. Removal of Antibody Complexes: (1) Option (Retrieval/Stripping 2-in-1 Buffer): Heat the working solution of Retrieval/Stripping 2-in-1 Buffer in a microwave at high power until boiling, hold for 10 seconds. Turn off the microwave and allow the solution to stand for 5 minutes. Transfer the beaker to a water bath to cool to room temperature. Used buffer is not recommended for reuse. (2) Traditional Method: Immerse slides in citrate buffer or EDTA antigen retrieval buffer. Heat in a microwave at high power until boiling, hold for 20 seconds, then switch to low power to sustain gentle boiling for 10-20 minutes. Place the retrieval container in a water bath to cool to room temperature.
13. Rinse with PBS Buffer for 5 minutes.
14. Repeat Steps 6-13 until all targets in the multi-round staining are completed.
15. Nuclear Staining: Apply an appropriate volume of nuclear dye to cover the tissue section. Incubate in a light-protected humidified chamber at room temperature for 5 minutes. Rinse 3 times with PBS Buffer for 5 minutes each.
16. Mounting: Apply 1-2 drops of Rapid Mounting Medium to cover the tissue section. Gently place a coverslip, transfer slides to a 37℃ incubator for 30 minutes ~ 1 hour. Note: Do not place in a humidified chamber.
17. Section Preservation: Place the sections in a slide box for long-term storage at -20℃. If stored at RT, keep them protected from light and complete imaging within 1 month.

Fluorophore Spectral Data

Note

Please cite this product as "IHCT003, Bioss Antibodies". Example: “Co-staining of A and B was performed using the Multiplex Fluorescent IHC Staining Kit (IHCT003, Bioss Antibodies), which relies on the Tyramide Signal Amplification (TSA) technology, following the manufacturer’s instructions.”

For research use only. Not for use in diagnostic or therapeutic procedures.