Multiplex Fluorescent (7 colors) IHC Staining Kit

Cat. No.: IHCT005
Detection method: Fluorescent
Section type: FFPE & Frozen Tissue, cells
Size: 20T | 100T
Storage and Stability: Store protected from light at 2–8℃ for 12 months.

Product Components

TSA fluorophore working solution = TSA concentrated fluorophore + TSA buffer.
The dilution ratio should be optimized according to the specific experiment. The recommended range is 1:50 to 1:400, and 1:200 gives the best results in most cases.

• If the primary antibody is incubated at room temperature for 1–3 hours, a dye dilution of 1:50–1:200 is recommended.
• If the primary antibody is incubated overnight at 4°C, a dilution of 1:200–1:400 or higher is recommended.
• If the signal is too strong, reduce the dye concentration, reaction time, or primary antibody concentration.
• If the signal is too weak, increase the dye concentration, reaction time, or primary antibody concentration.

PRINCIPLE

Tyramide Signal Amplification (TSA) is an enzymatic detection method that uses horseradish peroxidase (HRP) to label target proteins in situ. The principle is based on the peroxidase reaction of tyramide. Under the action of HRP and H₂O₂, the tyramide fluorophore substrate becomes activated. The activated fluorophore then covalently binds to tyrosine and other residues on the target protein, causing substantial fluorophore deposition at the antigen–antibody binding site and thereby amplifying the signal.

After each staining round, the previously bound non-covalent antibodies can be removed by heat-induced retrieval or an antibody elution buffer, while the fluorophore remains stably attached to the protein. This enables the next round of staining. Once all antibody incubations are completed, nuclear staining, mounting, and scanning are performed.

Because only one antibody system is present in each staining round, there is no need to worry about antibody cross-reactivity or host-species matching between primary and secondary antibodies. This overcomes the species-origin limitations common in traditional immunofluorescence experiments.

The fluorophores in this kit may be used individually or in combination and can support single staining, double staining, triple staining, and even more multiplex fluorescence labeling.

EXPERIMENTAL PROCEDURE

1. Sample Preparation

1) Paraffin sections

Place slides sequentially into:

• Xylene I, 15 min
• Xylene II, 15 min
• Absolute ethanol I, 5 min
• Absolute ethanol II, 5 min
• 95% ethanol, 5 min
• 85% ethanol, 5 min
• 75% ethanol, 5 min

Then rinse with distilled water.

2) Frozen sections

Fix frozen sections for 10–30 min, wash with PBS for 5 min × 3, add 0.3% Triton X-100 permeabilization solution for 20 min, then wash with PBS for 5 min × 3.

3) Cell climbing slides or cell smears

Fix cell samples for 10–30 min, wash with PBS for 5 min × 3, add 0.3% Triton X-100 permeabilization solution for 20 min, then wash with PBS for 5 min × 3.

2. Antigen Retrieval

Place tissue sections into a retrieval container filled with either:

• pH 9.0 EDTA alkaline antigen retrieval solution, or
• pH 6.0 citrate retrieval buffer

Perform antigen retrieval in a microwave oven (other methods such as pressure cooker 1–2 min, boiling water at 100°C for 15 min, or water bath at 95°C for 20 min may also be used).

Microwave program:

• medium heat for 8 min
• stop heating and stay still for 8 min
• medium-low heat for 7 min

During this process, prevent excessive evaporation of the buffer and do not allow the slides to dry out.

After natural cooling, place slides into PBS (pH 7.4), and wash on a shaker 3 times for 5 min each.

The choice of retrieval buffer and conditions depends on tissue type, fixation method, and antigen type. This step may be omitted for frozen sections and cell samples.

3. Block Endogenous Peroxidase

Incubate the slides in 3% H₂O₂ solution at room temperature, protected from light, for 15 min. Then wash in PBS (pH 7.4) on a shaker 3 times for 5 min each.

4. Block Non-specific Binding

After gently removing excess liquid, draw a hydrophobic barrier around the tissue with a PAP pen.

Add antibody diluent (or another blocking reagent such as 3% BSA or goat serum) to completely cover the tissue and block at room temperature for 30 min.

Note: The antibody diluent contains various stabilizers and preservatives and can be used for blocking or for dilution of primary antibodies. Diluted primary antibodies can be stored long-term at 4°C and for up to one month at room temperature.

5. Primary Antibody Incubation

Gently remove the blocking solution and add the appropriately diluted primary antibody working solution to the slide. Incubate flat in a humidified chamber protected from light:

• overnight at 4°C, or
• 1–2 h at 37°C

Add a small amount of water into the humid chamber to prevent antibody evaporation.

1. Secondary Antibody Incubation

Wash slides in PBS (pH 7.4) on a shaker 3 times for 5 min each. After gently removing excess liquid, add the HRP-conjugated secondary antibody corresponding to the host species of the primary antibody to fully cover the tissue. Incubate at room temperature for 50 min, protected from light, then wash with PBS 3 times for 5 min each.

Note: The kit includes a ready-to-use HRP goat anti-rabbit/mouse universal secondary antibody with very high sensitivity. No preparation is required.

2. Fluorescence Staining

Add the diluted TSA fluorophore working solution to completely cover the tissue and incubate at room temperature for 1–15 min.

Recommended reaction time: 5–10 min

Then wash with PBS 3 times for 5 min each.

Note: In a pilot experiment, stain for 1 min first, wash, and observe the result. If the positive signal is weak, continue applying the fluorophore reaction solution until suitable intensity is achieved before moving to the next step.

3. Antibody Elution

For paraffin sections, place slides into antigen retrieval solution and incubate in a 95°C water bath for 25–40 min (adjust the time according to antibody affinity), or use mIHC-specific antibody stripping buffer (Cat#C2208) as follows:

• apply an appropriate amount of prewarmed (37°C) mIHC-specific antibody stripping buffer to cover the sample
• incubate at 37°C for 5–20 min
• discard the stripping buffer
• apply fresh stripping buffer again
• incubate at 37°C for 5–20 min
• discard the stripping buffer
• wash with PBS 3 times for 5 min each

Note:

• Paraffin sections may use either heat retrieval elution or mIHC-specific antibody stripping buffer.
• Cells (cell climbing slides / cell smears), frozen sections, and fragile bone tissue must use the mIHC-specific antibody stripping buffer.

4. Second-round Labeling

Switch to another TSA fluorophore working solution and repeat Steps 3–7.

5. DAPI Nuclear Counterstaining

Wash slides in PBS (pH 7.4) on a shaker 3 times for 5 min each. After gently removing excess liquid, add ready-to-use DAPI staining solution within the marked area and incubate at room temperature for 5–20 min, protected from light.

6. Mounting

Wash slides in PBS (pH 7.4) on a shaker 3 times for 5 min each. After gently removing excess liquid, mount with the anti-fade mounting medium.

7. Microscope and Imaging

Observe and acquire images using:

• fluorescence microscope
• confocal microscope
• multichannel fluorescence scanner
• multispectral imaging system

PRECAUTIONS

1. Light protection

The fluorophores in this kit have strong anti-photobleaching properties. Full protection from room light is NOT required during the workflow, and operation in complete darkness is unnecessary. However, do not expose the reagents or samples to direct sunlight.

2. Causes of channel crosstalk

• Related to the filter bandwidth of the imaging equipment — use narrower bandwidth filters whenever possible.
• The antibody from the previous round was not completely eluted — optimize elution conditions for high-affinity antibodies, such as increasing elution temperature or time.
• Signal imbalance — if two adjacent channels have dyes where one signal is too strong and the other too weak, the stronger signal may bleed into the weaker channel.

3. Choice of antigen retrieval / antibody elution conditions

Recommended:

• First round antigen retrieval: pH 9.0 EDTA, 95°C for 15–25 min
• Second and subsequent rounds antigen retrieval / antibody elution: pH 6.0 citrate, 95°C for 25–40 min

For tissues that detach easily from the slide, antibody elution buffer may be used instead, but time and temperature must be controlled carefully. Excessive elution time or overly high temperature may reduce antigen recognition or weaken DAPI nuclear staining.

4. Biomarker / antibody order selection

• In the first round, choose markers suitable for pH 9.0 EDTA retrieval.
• Stain the more difficult markers in the earlier rounds.
• Antibodies that are difficult to elute should be used in the last round to avoid crosstalk.

5. Causes of non-specific staining

1. Polyclonal antibodies are more likely to produce non-specific staining; switch to monoclonal antibodies or reduce concentration and retrieval intensity.
2. Signal amplification may be too strong; reduce fluorophore reaction time or concentration.
3. Primary antibody concentration may be too high or retrieval may be excessive; use a higher antibody dilution or lower retrieval strength such as temperature, time, or buffer pH.

Notes

Please cite this product as "IHCT005, Bioss Antibodies". For example: ‘Co-staining of A and B was performed using the Multiplex Fluorescent IHC Staining Kit (IHCT005, 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.