Multiplex immunohistochemistry

Multiplex immunohistochemistry (mIHC), also known as tyrosine signal amplification (TSA), enhances the amount of observable information in a single tissue section by simultaneously staining it with multiple immune markers. Therefore, it serves as a powerful tool for directly visualizing cell interactions within the tissue microenvironment^[1].
Technical Principle: mIHC utilizes different fluorescent dyes to label tissues or cells through multiple rounds of staining, achieving multi-target staining. The HRP on the secondary antibody catalyzes the addition of inactive fluorescein TSA to generate an activated fluorescent substrate. This activated substrate covalently binds to the tyrosine residues of the antigen, covalently attaching the signal to the antigen. The fluorescence signal generated by this molecular binding lasts longer than that generated by traditional immune binding. Subsequent anti-repair washing removes the non-covalently bound antibodies, exposing the antigen for the next round of binding. After all antibody incubation is complete and the fluorescein has fully bound, the results are detected^[2].
Multiplex immunohistochemistry experiments are complex and require not only high-quality antibodies but also experienced operating skills. MCE provides you with high-quality mIHC-specific reagents and professional technical support services. For more service information, please visit the official website: https://www.medchemexpress.com/

MCE has not independently verified the accuracy of these methods; they are for reference only.

1. Sample preparation

(1) Tissue collection: When collecting tissue samples from animals or humans, special attention must be paid to controlling the volume of the tissue block to ensure it is within an appropriate range (containing more than 1,000 cells) so that the fixative can penetrate the tissue evenly and completely.
(2) PBS/Saline rinsing: Remove blood or other bodily fluids from the tissue sample that may affect the fixation effect.
(3) Fixation: Fix with 10% formalin, methanol, ethanol, or a mixture thereof at room temperature for 18-24 hours. Ensure the tissue sample is completely immersed in the fixative to avoid unfixed areas.
(4) Post-fixation treatment: Rinse off the fixative with running water.

2. Embedding and Sectioning

Common embedding media include paraffin and resin. Taking paraffin as an example:
(1) Dehydration: Dehydrate using an alcohol gradient, soaking the sample in 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol (I), and anhydrous ethanol (II) for 30-60 min each. The sample will become opaque during this process.
(2) Clearing: Soak the sample twice in a clearing agent (such as xylene), 30 min each time, to remove alcohol and other solvents. Avoid excessive clearing, which can cause the sample to harden and become brittle.
(3) Paraffin impregnation: Immerse the cleared tissue sample in molten paraffin and place it in a constant temperature incubator to allow the paraffin to penetrate fully and evenly into the tissue.
(4) Embedding: Place the paraffin-impregnated tissue sample into a mold and pour in molten paraffin. Allow the paraffin to cool and solidify, avoiding excessive cooling which can cause the paraffin to crack or the tissue to deform.
(5) Slicing: Cut the embedded tissue sample into 4-5 μm thick slices using a microtome.
(6) Spreading and baking: Gently separate the slices from the microtome with a fine brush, then place them in 40°C warm water to fully spread them out, and finally bake them at 60°C for 2 hours to enhance the adhesion of the slices.

3. Dewaxing and Antigen Retrieval

(1) Dewaxing: Dewaxing is performed sequentially using xylene I, II, and III, with each stage lasting 5 min. Thorough removal of paraffin is crucial for subsequent experiments; any residue will interfere with antigen exposure and staining.
(2) Rehydration: Dehydration is performed sequentially using a gradient of ethanol (100% ethanol I, 100% ethanol II, 95% ethanol, 85% ethanol, 70% ethanol), with each stage lasting 5 min. Finally, the slices are rinsed with purified water for 5 min to complete the hydration process. It is important to avoid drying the slices, as this can lead to high background noise from non-specific antibody binding.
(3) Antigen Retrieval: Antigen retrieval techniques expose blocked antigenic epitopes to enhance antibody binding efficiency. Heat-induced antigen retrieval is the most common method. After heating, the slices should be allowed to cool naturally. The following retrieval buffers are recommended: citrate buffer (pH 6.0), EDTA buffer (pH 8.0), or Tris-EDTA buffer (pH 9.0). EDTA and Tris-EDTA buffers can be used when citrate buffer is ineffective, but caution should be exercised as they may produce non-specific staining in high-expression samples. Several remediation methods can be tried to achieve the best staining results.

4. Blocking and Encapsulation

(1) Blocking: Incubate the sample with 3% H2O2 solution at room temperature in the dark for 15 min to eliminate endogenous peroxidase activity and avoid its interference with the subsequent HRP colorimetric system.
(2) Washing: Wash the slices twice with ddH2O for 5 min each time. Wash once with PBS on a shaker for 5 min each time.
(3) Encapsulation: Circle the sample area on the slice with a histochemical pen, add blocking solution, and incubate with blocking solution (5% BSA or serum) at room temperature or 37°C for 30 min.
(4) Washing: Wash three times with PBS on a shaker for 5 min each time.

5. Antibody incubation

(1) Primary Antibody Incubation: Select a primary antibody with high specificity and strong affinity according to the experimental purpose. The primary antibody should match the species, source, and type of the target antigen. Dilute the primary antibody to an appropriate ratio using a diluent (PBS, TBS, etc.). Use a pipette to evenly cover the sample area with the diluted primary antibody and place it in a humidity-balanced incubation box. Incubate at 37°C for 90 min to promote specific antigen-antibody binding.
(2) Washing: Wash three times with PBS on a shaker, 5 min each time. For samples prone to detachment, use a mild elution buffer pre-warmed to 37°C and wash gently for 5-20 min to maintain tissue integrity while effectively removing non-specific binding.
(3) Secondary Antibody Incubation: The secondary antibody is selected based on the type of primary antibody and sample characteristics. The role of the secondary antibody is to bind with the primary antibody to form an antigen-antibody-antibody complex, thereby enhancing the signal intensity and specificity. Dilute the secondary antibody to an appropriate ratio using a diluent (PBS, TBS, etc.). Incubate the secondary antibody at room temperature for 1 hour, taking care to avoid light and dry the slice.
(4) Washing: Remove the working solution of the secondary antibody and wash slowly with PBS on a shaker 3 times, 5 min each time, to remove unbound antibodies.

6. TSA Dye Incubation

(1) Diluting TSA: Dilute the TSA dye with 1×TSA buffer at a ratio of 1:50-1:200 to prepare a working solution. Store the staining solution at room temperature, protected from light, for up to 24 hours.
(2) Incubation: Add 100 μL of the staining working solution to a glass slide, immersing the sample. Incubate on a shaker at room temperature for 5-10 min, taking care to avoid drying.
(3) Washing: Wash three times with PBS on a shaker, 5 min each time.

7. Antibody Elution

(1) For frozen sections/cell smears/bone tissue (easy to elute): It is recommended to add an appropriate amount of preheated (37°C) mIHC-specific antibody elution buffer until completely dissolved, evenly covering the sample. Incubate at 37°C for 5-20 min, discard the elution buffer, and add another appropriate amount of antibody elution buffer to evenly cover the sample again. Incubate at 37°C for 5-20 min.
(2) Washing: Wash once with ddH2O, then soak in PBS for 2 min.

8. Repeat Staining

Continue staining, starting from step "Blocking". Repeat the steps including blocking and encapsulation—antibody incubation—TSA dye incubation—antibody elution.

9. Nuclear Counterstaining

After all staining are completed, stain the cell nuclei with a staining agent to visually distinguish the location of the cell nucleus from other cells and antigen structures, facilitating observation and interpretation of experimental results.
(1) Add DAPI/ Hoechst working solution to the sample, incubate at room temperature for 5 min, and wash once with PBS.

10. Mounting and Observation

(1) Mounting: Slowly and evenly drop an appropriate amount of anti-fluorescence quenching mounting solution onto the slice. Use tweezers to gently place the coverslip onto the slice, avoiding air bubbles. The cell side should be close to the slice. Use absorbent paper to remove excess mounting material. Gently seal the coverslip with nail polish to prevent the sample from moving under the microscope. After the nail polish dries completely, observe directly or store at 4°C in the dark.
(2) Observation: Observe the slice under a fluorescence microscope/confocal microscope/multichannel fluorescence scanner/multispectral imaging system and acquire images.

for high-expression primary antibodies and high-light for low-expression primary antibodies. It is recommended to assess the staining efficacy of each antibody through preliminary experiments before performing multiplex immunostaining. Prioritize labeling target antigens with weaker staining signals, and then stain other antigens sequentially.
2. Antigen Retrieval: It is recommended to use an excess of antigen retrieval buffer. No single antigen retrieval buffer is suitable for all antigens. Different antibodies may require different antigen retrieval methods and conditions; therefore, the appropriate antigen retrieval method should be selected based on experimental requirements and antibody characteristics.
3. HRP Blocking: H2O2 should be prepared fresh and stored at 4°C in the dark; otherwise, non-specific background may occur. Prolonged H2O2 incubation can also cause slide detachment. Some tissues also contain endogenous alkaline phosphatase, which can be inactivated with levamisole.
4. Protection from Light and Avoiding Drying: Multiplex staining is a lengthy process; care should be taken to protect the slides from light to prevent fluorescence quenching. During the experiment, maintain a moist state to avoid drying the slides, which can easily lead to high background or uneven imaging.
5. Formalin-fixed and paraffin-embedded samples should ensure the integrity of the tissue block, without visible cracks or structural damage. Tissue microarray (TMA) blocks must maintain the integrity of the embedding matrix, avoiding cutting or sealing defects. Sections on glass slides should show continuous and complete tissue structures, without folds or fragmentation.
6. TSA fluorescent dye working solution = VF fluorescent dye + TSA buffer; the recommended dilution ratio of fluorescent dye to TSA buffer is 1:200; the dilution ratio can be flexibly adjusted and optimized according to specific circumstances, with an optimal range of 1:50-1:400; generally, if the primary antibody incubation time is within 1-3 hours at room temperature, a dilution ratio of 1:50-1:200 is recommended; if the primary antibody incubation time is overnight (12 hours or more) at 4°C, a dilution ratio of 1:200-1:400 or higher is recommended.
7. When using several fluorescein, choose those with no spectral overlap to reduce the labeled fluorescence intensity. High fluorescence intensity in one or more samples can sometimes lead to spectral overlap. Reducing the label concentration, shortening the labeling time, and adjusting the fluorescein medium can lower the sample fluorescence intensity. 8. Sealed slides can be stored long-term, but should be kept in a dry, dark environment at a suitable temperature to prevent moisture absorption, fading, or damage.