Multiplex Immunohistochemistry Protocol

Licia Miller   Product Manager

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Multiplexed immunohistochemistry (mIHC) is an in situ multiple labeling method based on tyramide signal amplification (TSA) technology, which can achieve simultaneous detection of multiple targets (usually 6-8 types) on a single tissue section. Its core principle is to covalently bind the target antigen through HRP-catalyzed fluorescent tyramide substrate, and then remove the non-covalently bound antibody complex through heat repair or antibody elution solution, and replace antibodies and fluorescent dyes in rounds to complete multiple rounds of labeling. This technology is widely used in tumor microenvironment analysis, immune cell spatial localization, drug target evaluation and other fields.

Experimental Steps

1. Sample Preparation

Dewaxing and hydration of paraffin sections

1.1 Immerse the sections in xylene three times for 10 minutes each time to remove the paraffin from the sections.

1.2 Place the slices in anhydrous ethanol and repeat twice, 10 minutes each time.

1.3 Soak in gradient ethanol (95%, 85%, 75%) for 10 minutes each.

1.4 Wash the sections with dH2O twice, 5 minutes each time.

Dosage: At each step, immerse the sections in liquid volume sufficient to cover the tissue (approximately 50-100 mL/slide).

Frozen section/cell slide processing

1.1 Soak the slices/slides in 4% paraformaldehyde for 10-30 minutes for fixation.

1.2 Soak in 0.3% Triton X-100 and permeabilize the membrane for 20 minutes.

1.3 Rinse with PBS 3 times, 5 minutes each time.

2. Antigen Retrieval

Microwave repair

2.1 Using a microwave oven, place the slides in pH 6.0 10 mM sodium citrate buffer ( sodium citrate 2.94 g/L or EDTA 0.37 g/L + Tris 1.21 g/L ) and bring to a boil on high heat.

2.2 Stop heating and keep the slide at residual temperature for 10 minutes.

2.3 Continue heating over medium-low heat for 7 minutes (pay attention to replenishing liquid to prevent excessive evaporation and causing dry slices).

2.4 Take out the slide and place it on the table to cool to room temperature for 30 minutes. This will open up the protein epitopes, making it easier for the antibody to bind to the target antigen and improve the staining effect.

High voltage repair

Boil in 100℃ water for 15 minutes or in a water bath for 20 minutes.

3. Blocking Endogenous Peroxidase

3.1 Wash the sections 3 times with dH2O, 5 minutes each time.

3.2 Soak the slices in 3% Incubate in hydrogen peroxide solution at room temperature in the dark for 15 minutes to block endogenous peroxidase.

3.3 Wash the sections 3 times with 1× TBST, 5 minutes each time.

3.4 After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), add 3% BSA or normal goat serum to evenly cover the tissue, and block at room temperature for 30 minutes.

4. Antibody Incubation​

4.1 Prepare the primary antibody diluent and dilute the primary antibody according to the recommended dilution of the antibody.

4.2 Add the diluted primary antibody to the slice sample, usually 100-400 μl per slice, and then place the slice in a humidified chamber at 4℃ overnight or at room temperature for 1-2 hours.

4.3 Remove the primary antibody solution and replace with 1× TBST Rinse gently 3 times, 5 minutes each time.

4.4 drops of poly-HRP labeled secondary antibody dilution solution to the sliced tissue and incubate in a humidified box at room temperature in the dark for 1 hour.

4.5 Remove the secondary antibody solution and replace with 1× TBST Rinse gently 3 times, 5 minutes each time, protecting from light.

5. TSA Fluorescent Dye Reaction

5.1 Select appropriate fluorophore-conjugated TSA reagents based on the expression level of the target.

5.2 Dilute the reagent according to the recommended concentration of TSA reagent.

5.3 Add 100-400 μl of diluted TSA reagent to the sliced tissue and incubate it in a humidified box at room temperature for 5-10 minutes in the dark. Be careful to avoid light.

5.4 Use 1× TBST rinse gently 3 times for 5 minutes each, protecting from light.

6. Repeat Staining (Multiple Rounds of Labeling)

If you want to detect multiple targets for multiple staining, you can jump directly to step 7 for antibody stripping and re-staining;

If you have completed multiplex staining or are performing singleplex staining, you can proceed directly to step 8 for coverslipping.

When multiple rounds of labeling are performed, the primary antibody and TSA dye (different excitation wavelength) are changed in each round. The recommended staining order is: low-abundance target → high-abundance target, weak fluorescent dye → strong fluorescent dye.

7. Antibody Stripping

7.1 For paraffin sections, use preheated antigen retrieval solution and place in a 100℃ water bath for 25-40 minutes.

For frozen/cell samples , use mIHC dedicated elution buffer , treat at 37℃ for 5-20 minutes, repeat twice.

7.2 After elution, rinse with PBS three times, 5 minutes each time.

8. DAPI Counterstaining and Sealing

8.1 Add DAPI stain to the slices and incubate at room temperature in the dark for 10 minutes.

8.2 Wash the slides 3 times with 1× TBST buffer and place at room temperature for 3 minutes each time.

8.3 Use a pipette to add antifade mounting medium onto the slide, making sure to immerse the sample area.

8.4 Cover with a coverslip, seal the edges of the slide with nail polish, and store away from light or use directly for microscopic observation.

Key Considerations

(1) Antibody optimization: The primary antibody concentration and TSA dye exposure time should be determined through single-label pre-experiments (recommended exposure time 50-200 ms, channel-to-channel difference ≤ 10 times).

(2) Elution control: Insufficient elution will lead to residual signal interference, while excessive elution may destroy the antigen.

(3) Autofluorescence processing: The wavelength of tissue autofluorescence is recorded through spectral imaging to avoid overlap with the target signal.

(4) The experimental design needs to be based on the target expression abundance, antibody affinity and spectral characteristics of the fluorescent dye. It is recommended to refer to the optimization cases in the literature.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/