mIHC-specific Antibody Stripping Buffer

Cat. No.: C2208
Size: 30mL
Storage and Stability: Store at room temperature for 12 months.

Application: Suitable for paraffin sections, frozen sections, cell climbing slides, and cell smears for antibody stripping during TSA staining workflows 

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
• 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.

    6. 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.

     7. 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.

     8. Antibody Stripping

Add an appropriate amount of mIHC Antibody Stripping Buffer, prewarmed to 37°C until fully dissolved, to cover the sample (recommended for cell climbing slides, cell smears, frozen sections, and bone tissue prone to detachment).

• Incubate at 37°C for 5–20 min 

• Discard the stripping buffer 

• Add fresh stripping buffer again to cover the sample 

• Incubate at 37°C for 5–20 min 

• Discard the stripping buffer 

• Wash with PBS for 5 min, repeat 3 times 

Note:

• Paraffin sections may use either heat retrieval stripping or stripping buffer.
• Cells and frozen sections must use the mIHC-specific antibody stripping buffer.

     9. Second-round Labeling

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

     10. 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.

      11. 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.

      12. Microscope and Imaging

Observe and acquire images using:

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

PRECAUTIONS

1. The stripping efficiency depends on section thickness, stripping temperature and time, and the type of primary antibody. The exact stripping time should be adjusted according to the specific situation. 
2. If antibody stripping is problematic, you may appropriately reduce the primary antibody concentration or place those antibodies in the final staining round 2.
3. The antibody stripping buffer can flow off the sample easily, so if runoff is observed it should be replenished promptly. 
4. Before each use, ensure the reagent is fully dissolved with no precipitate. If precipitation is present, place it at 37°C until fully dissolved before use. 
5. The mIHC-specific antibody stripping buffer is primarily recommended for cell climbing slides, cell smears, frozen sections, and bone tissue prone to section loss. For paraffin sections, EDTA or citrate retrieval buffer is preferred for antibody stripping. 
6. The mIHC-specific antibody stripping buffer is slightly acidic. Over-stripping (too long or too high a temperature) may lead to reduced antigen recognition and weaker DAPI nuclear staining. 
7. Unfixed sections are not suitable for use with this mIHC-specific antibody stripping buffer. 

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