Multiplex Immunofluorescence Experimental Procedure + Precautions

In recent years, Multiplex Immunohistochemistry (mIHC), with its ability to simultaneously detect multiple markers and analyze spatial information, has become an important tool for tumor microenvironment research, immunotherapy evaluation, and spatial pathology studies. However, compared to traditional IHC, the mIHC experimental workflow is more complex. From antibody screening to Panel design, to TSA staining and image analysis, every step can affect the final result.

This article will guide you through the TSA-mIHC experimental workflow and highlight common problems and solutions encountered in the process.

Currently, mIHC most commonly uses FFPE (Formalin-Fixed Paraffin-Embedded) tissue samples. Tissue section thickness is typically controlled at 4-5 μm.

Baking: Gently separate the sections from the knife using a fine brush, then place them in 40°C warm water to flatten completely, and finally bake at 60°C for 2 hours to enhance section adhesion. Bake at 60°C for 1 hour to ensure the tissue is firmly attached to the slide.

Precautions

✓ Use adhesive slides

✓ Avoid tissue folding

✓ Fresh sections are preferable to long-term stored sections

Purpose: Remove paraffin and gradually restore the hydrated state of the tissue, preparing for subsequent antigen retrieval.

Common procedure: Xylene I (10 min) → Xylene II (10 min) → Absolute ethanol (5 min) → 95% ethanol (5 min) → 85% ethanol (5 min) → 75% ethanol (5 min) → Distilled water wash

Common problem: Tissue edge detachment

Possible causes:

      • Insufficient baking

      • Excessive deparaffinization time

      • Insufficient slide adhesion

Formalin fixation causes protein crosslinking, masking antigen epitopes, so antigen retrieval must be performed.

Heat-induced antigen retrieval is the most commonly used method. Common buffers include: Citrate buffer (pH 6.0) and EDTA buffer (pH 8.0-9.0). EDTA buffer can be used when citrate buffer yields poor results, but attention should be paid to potential non-specific staining in high-expression samples. Multiple retrieval methods can be attempted to achieve optimal staining results.

Microwave retrieval: Add antigen retrieval solution, place the section in a microwave oven at medium power for 8 min, pause for 7 min, then switch to low power for 8 min; remove the staining box and cool to room temperature.

Water bath retrieval: Add antigen retrieval solution, place the section in a water bath at 95°C for 25-30 min (note temperature control to avoid boiling to prevent tissue detachment). Cool the section with water gradually, do not cool rapidly, to avoid conformational changes in antigen epitopes due to rapid temperature decrease.

Pressure cooker retrieval: Add antigen retrieval solution to the staining box, place the section, heat in a pressure cooker until saturated pressure is reached, continue heating for 5 min, turn off power, remove the staining box after 10 min, and cool to room temperature.

Enzymatic retrieval: Commonly used digestive enzymes include trypsin, pepsin, proteinase K, etc. Place the section on a slide containing enzymatic solution, then perform enzymatic digestion under appropriate temperature and humidity conditions (typically 37°C, 20 min). After enzymatic treatment, cool the section to room temperature before proceeding. After retrieval, wash 3 times with PBS on a shaker, 5 min each time.

Precautions

✓ Different antibodies may have completely different optimal retrieval conditions.

✓ It is recommended to complete single staining validation first.

Incubate the sample with 3% H₂O₂ solution at room temperature, protected from light, for 20 min to eliminate endogenous peroxidase activity and avoid interference with subsequent HRP detection systems.

Washing: Rinse the slide twice with ddH₂O, 5 min each time. Wash once with PBS on a shaker, 5 min each time.

Use a histology pen to circle the sample area on the slide, add blocking solution to reduce non-specific binding background. Incubate at 37°C for 30 min.

Common reagents:

      • 3% H₂O₂ (block endogenous peroxidase)

      • Normal goat serum

      • BSA blocking solution

Washing: Wash 3 times with PBS on a shaker, 5 min each time.

Select an appropriate primary antibody and dilute it to a suitable ratio using diluent (PBS, TBS, etc.). Use a pipette to evenly cover the sample area with the diluted primary antibody, place in a humidity-balanced incubation box, and incubate at 37°C for 1 hour or overnight at 4°C. Recommendation: Prioritize antibodies validated for IHC.

Washing: Wash 3 times with PBS on a shaker, 5 min each time. For sections prone to detachment, use gentle wash buffer pre-warmed to 37°C and wash gently for 5-20 minutes to maintain tissue integrity while effectively removing non-specific binding.

Antibody Selection Principles

✓ Clear localization

✓ Good specificity

✓ Sufficient literature support

✓ FFPE validation passed

Select an appropriate HRP-labeled secondary antibody and dilute it to a suitable ratio using diluent (PBS, TBS, etc.). Incubate at 37°C for 1 hour, protecting from light and keeping the slide moist.

Washing: Wash 3 times with PBS on a shaker, 5 min each time.

This is the most critical step in TSA-mIHC.

Add 100 μL of staining working solution to the slide, immerse the sample, and incubate at 37°C for 30 min. HRP catalyzes the deposition of fluorescence-labeled tyramide molecules around the antigen, forming covalently bound fluorescent signals.

Washing: Wash 3 times with PBS on a shaker, 5 min each time.

Features:

✓ High sensitivity

✓ Signal amplification

✓ Signal is not easily lost during subsequent retrieval

Add antibody stripping solution to the tissue and incubate at 37°C for 15 min.

Purpose: Remove the current round of antibodies while retaining the deposited fluorescent signal, allowing the next round of staining to proceed.

Starting from "Blocking", repeat the steps: Blocking → Primary Antibody Incubation → HRP Secondary Antibody Incubation → TSA Fluorescence Deposition → Antibody Stripping.

Complete detection of 2-6 markers, or even more markers, according to the Panel design sequence.

Empirical principles:

✓ High-expression antigens should be stained first

✓ Weak-expression antigens should be stained later

✓ Avoid signal overlap

After completing all markers, add DAPI working solution to the sample and incubate at room temperature for 5 min. Wash 3 times with PBS, 5 min each time. Drain the liquid, add anti-fade mounting medium to the tissue, and cover with a coverslip.

Store the prepared fluorescent slide at 4°C protected from light.

Functions:

✓ Cell counting

✓ Cell segmentation

✓ Basis for spatial analysis

Observe and acquire images using a fluorescence microscope/confocal microscope/multichannel fluorescence scanner/multispectral imaging system.

Complete the following using professional software: Spectral unmixing, cell segmentation, phenotyping analysis, spatial analysis, etc.