Multiplex Immunohistochemistry (mIHC) Operation Procedure

1. Sample Pretreatment

Slice Baking：Place paraffin/frozen sections in an oven set at 65°C for overnight baking (<16h).

Slice Rehydration：

a) Paraffin section dewaxing to water: Place sections sequentially in staining jars containing xylene-1, xylene-1', xylene-1", soaking for 5 minutes each. Subsequently, place sections sequentially in staining jars containing absolute ethanol-1, absolute ethanol-1', absolute ethanol-1", 95% ethanol, 85% ethanol, 75% ethanol, soaking for 3 minutes each. Finally, place sections in a staining jar containing purified water, replace with fresh purified water sequentially, wash 3 times, 3 minutes each time.

b) Frozen section rehydration: After frozen sections return to room temperature (21~25°C), place sections in a staining jar containing PBS solution, replace with fresh PBS solution sequentially, wash 3 times, 2 minutes each time.

Antigen Retrieval：If repair is needed, two repair methods can be used (or no repair can be selected according to sample conditions)

Microwave Repair：Add appropriate amount of 1×citric acid (PH6.0) buffer to a transparent beaker (adjust according to the number of sections), place tissue sections and put them in a microwave oven, heat on high power until boiling, about 3 minutes; cool at room temperature for 30 minutes, then rinse with running tap water to cool to room temperature.

Blocking Endogenous Peroxidase：Use an immunohistochemical pen to draw a water-resistant circle around the tissue to ensure that the reagents added later can completely and evenly cover the tissue and prevent drying. Place the sections with the drawn water-resistant circles flat in a moist box, add appropriate amount of endogenous catalase blocker to completely and evenly cover the tissue, incubate at room temperature in the dark for 10 minutes. Place the sections in a staining jar containing PBS solution, replace with fresh PBS solution sequentially, wash 3 times, 2 minutes each time.

2. First Marker Staining

Primary Antibody Incubation：

After diluting the antibody to the corresponding concentration with prepared primary antibody dilution buffer, place the sections flat in a moist box with the front side up, add appropriate amount of diluted primary antibody to completely and evenly cover the tissue, incubate at 28°C±2°C with shaking in the dark for 20-30 minutes.

Place the sections in a staining jar containing PBS solution, replace with fresh PBS solution sequentially, wash 3 times, 2 minutes each time.

Secondary Antibody Incubation：

According to the species of the primary antibody, select the corresponding species' [TSA-HRP (R)] or [TSA-HRP (M)], place the sections flat in a moist box with the front side up, add appropriate amount of [TSA-HRP (R)] or [TSA-HRP (M)] to completely and evenly cover the tissue, incubate at 28°C±2°C with shaking in the dark for 20-30 minutes.

Place the sections in a staining jar containing PBS solution, replace with fresh PBS solution sequentially, wash 3 times, 2 minutes each time.

TSA Color Reaction：

Dilute [TSA Diluent]: PBS solution at a ratio of 1:299, invert and mix about 3 times to obtain TSA Diluent working solution.

Continue to dilute Cyclic-480: TSA Diluent working solution at a ratio of 1:250-1:500.

Place the sections flat in a moist box with the front side up, add appropriate amount of diluted Cyclic-480 to completely and evenly cover the tissue, incubate at room temperature in the dark for 5-10 minutes.

Place the sections in a staining jar containing PBS solution, replace with fresh PBS solution sequentially, wash 3 times, 2 minutes each time.

Observe under a fluorescence microscope to check for specific staining, and proceed to the next marker staining after confirming specific staining.

Antibody Elution（According to needs, choose one of the following two methods）：

a）High Temperature Elution：

Dilute [Citrate Antigen Retrieving Buffer]: purified water at a ratio of 1:49, mix well and pour into a staining jar.

Place the sections in the staining jar, heat with maximum power in a microwave oven for 3±1 minutes, after the reagent in the staining jar shows small bubbles, transfer to a constant temperature water bath preheated to 95°C in advance, keep at 95°C or above for 10-15 minutes.

Take out the staining jar and place it on a shaker, cool naturally to room temperature.

Place the sections in a staining jar containing PBS solution, replace with fresh PBS solution sequentially, wash 3 times, 2 minutes each time.

b）Mild Elution：

Place the sections flat in a moist box with the front side up, add appropriate amount of preheated [1x Ab Stripping Buffer] at 60°C to completely and evenly cover the tissue, incubate at 28°C±2°C in the dark for 10-20 minutes.

Place the sections in a staining jar containing PBS solution, replace with fresh PBS solution sequentially, wash 3 times, 2 minutes each time.

Note: This method is only suitable for: antibodies labeled after elution, which have different species origins from previously labeled antibodies (excluding antibodies labeled before high-temperature elution steps).

3. Second Marker Staining

Perform the same operations as the first marker, replace the fluorescent molecule with [Cyclic-550], dilute [Cyclic-550] with [TSA Diluent working solution] at a ratio of 1:250-1:500, and develop for 5-10 minutes.

4. Third Marker Staining

Perform the same operations as the first marker, replace the fluorescent molecule with [Cyclic-630], dilute [Cyclic-630]: [TSA Diluent working solution] at a ratio of 1:100-1:250, and develop for 10-15 minutes.

5. Nuclear Staining

Dilute [DAPI]: PBS solution at a ratio of 1:49.

Place the sections flat in a moist box with the front side up, add appropriate amount of diluted [DAPI] to completely and evenly cover the tissue, incubate at room temperature in the dark for 5 minutes.

Place the sections in a staining jar containing PBS solution, replace with fresh PBS solution sequentially, wash 3 times, 2 minutes each time.

Observe under a fluorescence microscope to check for specific staining, and proceed to mounting after confirming specific staining.

6. Mounting

Use a rubber dropper to draw [Mounting Medium], add a drop of [Mounting Medium] to completely and evenly cover the tissue, use tweezers to pick up a clean coverslip of suitable size, gently cover the tissue, avoiding air bubbles.

7. Microscopic Imaging

Place the sections under a fluorescence microscope/confocal/multichannel fluorescence scanner/multispectral imaging system to observe and collect images.

Please refer to the following table for imaging wavelength settings.

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