IHC experimental procedures typically include sample fixation, embedding, sectioning, dewaxing and hydration, antigen retrieval, inactivation, blocking, primary antibody incubation, secondary antibody incubation, color development, counterstaining, and mounting for observation, a total of 12 parts.

 

1. Sample Fixation

 

Purpose: To ensure the quality and integrity of the tissue samples, laying a solid foundation for subsequent immunostaining.

Operational Steps:

1) Sampling: Obtain the required tissue samples from animals or humans. For tissue blocks, ensure that their size is moderate to allow the fixative to penetrate fully;

2) Preliminary Treatment: If the tissue samples are contaminated with blood or other body fluids, rinse them with saline or PBS to remove substances that may affect the fixation effect;

3) Fixation: Place the tissue samples into the fixative. Commonly used fixatives include 4% paraformaldehyde solution, and OCT embedding medium can be used for frozen sections/neural tissues. The volume of the fixative is usually 10-20 times that of the tissue to ensure full immersion. Large tissue specimens should be cut open for fixation to prevent autolysis and putrefaction in the center.

 Note: The fixation time varies depending on the tissue type and experimental requirements, generally at room temperature for 18-24 hours.

4) Post-Fixation Treatment: After fixation, remove the tissue from the fixative and rinse it with running water for several minutes to remove residual fixative and any crystalline products that may have formed.

Precautions:

The fixation time should not be too long to avoid excessive hardening of the tissue and loss of antigenicity;

The fixation temperature is usually at room temperature or 4°C to prevent tissue damage caused by high temperatures;

During fixation, ensure that the tissue samples are fully immersed in the fixative to avoid any un-fixed parts;

The fixation container should be clean and free of impurities to prevent contamination of the tissue.

 

2.Embedding

 

Purpose: To protect and support the tissue samples, maintaining their integrity during sectioning. Embedding the tissue samples into a solid medium allows the production of thin sections suitable for microscopic observation.

Depending on the sample type, it can be divided into paraffin-embedded immunohistochemistry (IHC-P) and frozen-section immunohistochemistry (IHC-F). Although IHC-P is more complex in operation, it better preserves the tissue morphology, allows for thinner sections that are easier to stain and observe, and can be stored at room temperature for many years, making it more commonly used in experiments.

Basic Steps:

1) Dehydration: After fixation, the water content in the tissue samples needs to be removed to allow the embedding medium to penetrate fully. Ethanol gradient dehydration: 75% ethanol (1h)——85% ethanol (1h)——95% ethanol (1h)——100% ethanol (50min)——100% ethanol (50min)——100% ethanol (50min)

2) Clarification: After dehydration, the tissue samples become opaque and need to be clarified using a clarifying agent (such as xylene) to remove ethanol and other solvents, making the tissue samples transparent. Typically, xylene is used for immersion: xylene (40min)——xylene (40min)——xylene (40min)

3)Paraffin Infiltration: Place the clarified tissue samples into molten paraffin to allow the paraffin to penetrate fully into the tissue samples. This process usually needs to be carried out in a warming box to ensure uniform penetration of the paraffin: 63℃ paraffin (50min)——63℃ paraffin (50min)——63℃ paraffin (50min)

4) Embedding: Place the paraffin-infiltrated tissue samples into a mold, pour molten paraffin over them, and then allow the paraffin to cool and solidify. In this way, the tissue samples are embedded in paraffin.

Precautions:

During embedding, control the temperature and time to ensure uniform penetration of the paraffin and the integrity of the tissue.

The embedded tissue blocks need to be cooled and solidified before sectioning. During cooling, avoid excessive cooling that may cause the paraffin to crack or the tissue to deform.

Before embedding, the tissue samples should be fully fixed and dehydrated to ensure their antigenicity and integrity.

 

3.Sectioning

 

Purpose: To cut the embedded tissue samples into thin sections for microscopic observation and detection.

Basic Steps:

1) Preparations Before Sectioning: Ensure that the embedded tissue blocks have been fully cooled and solidified. Use an appropriate microtome for sectioning;

2) Sectioning: Fix the embedded tissue block onto the sample holder of the microtome. Adjust the section thickness of the microtome, usually 4-5 micrometers thick, which is determined based on the antigen to be detected and experimental requirements;

3) Spreading: Gently remove the cut tissue sections from the knife with a brush or brush pen and place them in warm water (40℃) to spread;

4) Baking: Bake the sections at 60℃ for 2 hours.

Precautions:

During sectioning, control the speed of the microtome and the sharpness of the sectioning knife to obtain high-quality sections.

After sectioning, promptly process the sections to avoid antigen loss and tissue denaturation.

Use an appropriate adhesive to ensure that the sections are firmly attached to the slides for subsequent immunohistochemical experiments.

 

4.Dewaxing and Hydration

 

Purpose: To release the tissue sections from paraffin and restore them to a state suitable for subsequent antigen detection and staining.

Basic Steps:

1) Dewaxing: The sections are first immersed in xylene to dissolve and remove the paraffin from the sections. Place the sections in a slide rack: xylene (5min)——xylene (5min)——xylene (5min)

2) Hydration: 100% ethanol (5min)——100% ethanol (5min)——95% ethanol (3min)——85% ethanol (3min)——75% ethanol (3min)——deionized water (5min)

Precautions:

Thorough dewaxing: The dewaxing process must be thorough to ensure that the paraffin is completely removed. Otherwise, residual paraffin will affect subsequent antigen detection and staining.

Avoid drying: During the entire dewaxing and hydration process, ensure that the sections remain moist to avoid drying. Drying can lead to non-specific antibody binding, resulting in high background staining.

 

5.Antigen Retrieval

 

Purpose: To expose antigen epitopes hidden during fixation through the retrieval process, enhancing the efficiency of antibody binding. The most commonly used retrieval buffers are 10mM sodium citrate (pH 6.0), Tris-EDTA (pH 9.0), and EDTA (pH 8.0). It is recommended to test multiple methods to find the one that yields the best staining results.

Antigen Retrieval Methods:

1) Microwave Thermal Retrieval: Add the antigen retrieval solution, place the sections in it, and put them in a microwave oven on medium heat for 8 minutes, stop for 7 minutes, then switch to low heat for 8 minutes; remove the staining box and let it cool to room temperature;

2) Water Bath Thermal Retrieval: Add the antigen retrieval solution, place the sections in it, and put them in a water bath pot for heating. Continuously measure the temperature with a thermometer. Once the temperature of the antigen retrieval solution reaches the effective level (92℃), maintain it for 40 minutes, then remove the staining box and let it cool to room temperature;

3) High-Pressure Thermal Retrieval: Add the antigen retrieval solution to the staining box, place the sections in it, and put the box in a pressure cooker. Heat until the pressure is reached and continue heating for 5 minutes. Turn off the power and remove the staining box after 10 minutes, letting it cool to room temperature;

4) Enzymatic Digestion Retrieval: Commonly used digestive enzymes include trypsin, pepsin, and proteinase K. Place the sections on slides containing the enzymatic digestion solution, then digest them under appropriate temperature and humidity conditions (usually at 37°C for 20 minutes). After enzymatic digestion, the sections also need to be cooled to room temperature before further processing.

Precautions:

Allow natural cooling after heating;

Use an excess of antigen retrieval solution;

The pH value of the retrieval solution has a significant impact on the staining results;

There is no one-size-fits-all antigen retrieval buffer for all antigens. Different antibodies may require different antigen retrieval methods and conditions, so it is necessary to select the appropriate antigen retrieval method based on experimental requirements and antibody characteristics.

 

6.Inactivation

 

Purpose: To eliminate the effects of endogenous enzymes and active substances in cells or tissues, reducing background noise and improving the specificity and sensitivity of the experiment.

1)Inactivation of Endogenous Peroxidase: For HRP detection systems, the commonly used method to remove endogenous peroxidase is a 3% hydrogen peroxide solution, at room temperature for 10 minutes;

2)Washing: Wash the sections with buffers such as PBS or TBS, soaking them twice for 5 minutes each.

 Precautions:

H₂O₂ should be prepared fresh and stored at 4℃ in the dark, otherwise it may cause non-specific background;

Prolonged incubation with H₂O₂ may also cause sections to fall off;

Some tissues also contain endogenous alkaline phosphatase, which can be inactivated with levamisole.

 

7.Blocking

 

Purpose: To block non-specific binding sites on tissue sections, preventing antibodies from binding to these sites and thereby reducing background signals.

Basic Steps:

1) Blocking of Non-Specific Sites: Add the blocking solution to the tissue sections, ensuring that the blocking solution evenly covers the entire tissue area. Then place the sections in a humidified box and incubate at room temperature or 37°C for 30 minutes to allow the blocking solution to fully interact with the tissue sections.

2) Washing: After blocking, wash the sections with buffers such as PBS or TBS to remove unbound blocking solution and any possible impurities.

 

8.Primary Antibody Incubation

 

Primary antibody incubation in immunohistochemistry is one of the key steps in the experiment.

Purpose: To specifically recognize the target antigen and provide an accurate marker for subsequent detection.

Basic Steps:

1) Selection of Primary Antibody: Choose a primary antibody with high specificity and strong affinity based on the experimental objective. The primary antibody should match the species origin and type of the target antigen;

2) Dilution of Primary Antibody: Dilute the primary antibody according to its titer and experimental requirements using an appropriate diluent such as PBS, TBS;

3) Application of Primary Antibody: Evenly apply the diluted primary antibody to the blocked tissue sections to ensure that the antibody can fully contact the antigens on the tissue sections;

4) Incubation: Add the diluted primary antibody evenly to the blocked tissue sections, place them in a humidified box, and incubate at room temperature (or 37°C) for 1 hour to allow the primary antibody to fully bind to the antigen.

Precautions:

Ensure the quality and specificity of the primary antibody to avoid using expired or poor-quality antibodies;

Thoroughly wash the tissue sections to remove unbound primary antibodies and other impurities, reducing background staining.

 

9.Secondary Antibody Incubation

 

Purpose: The secondary antibody binds to the primary antibody, forming an antigen-antibody-antibody complex, thereby enhancing the intensity and specificity of the signal.

In immunohistochemical experiments, secondary antibodies usually carry labeled enzymes (such as HRP, AP, etc.), which can produce visible staining signals in the subsequent color development steps. These signals facilitate the observation of the distribution and expression of the target antigen in tissues under a microscope.

Basic Steps of Secondary Antibody Incubation:

1) Dilution of Secondary Antibody: Dilute the secondary antibody according to the instructions, using the blocking solution or another appropriate solution;

2) Incubation of Secondary Antibody: Add the diluted secondary antibody to the tissue sections that have been incubated with the primary antibody and washed, and incubate at room temperature for 30-60 minutes.

3) Washing: After incubation, wash the sections with a washing solution (such as PBST or TBST) on a rocker, to remove unbound secondary antibodies and other impurities. Washing usually needs to be repeated several times, with each wash lasting about 5-10 minutes.

 

10.Color Development Reaction

 

Purpose: Other factors to consider in color development detection include the selection of enzymes and color development substrates. Each detection enzyme has several different colorimetric agents, with HRP-DAB being the most commonly used combination.

Principle: In immunohistochemistry, enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) are commonly used as labeling enzymes. These enzymes can catalyze the color reaction of the substrate, forming visible precipitates at the location of the antigen.

Basic Steps:

1) Add the enzyme substrate (e.g., DAB for HRP, BCIP/NBT for AP).

2) The substrate undergoes a chemical reaction under the action of the enzyme, forming a colored precipitate, usually brown (for the HRP-DAB system) or blue (for the AP-BCIP/NBT system).

3) Closely monitor the degree of color development and the color development time of the sections, generally 3-8 minutes, and promptly wash with distilled water to prevent over-coloring. By observing the color changes of the tissue sections under a microscope, the location and expression level of the antigen in cells or tissues can be determined.

Precautions:

Prepare DAB working solution fresh

Select the appropriate detection system: Choose the detection system based on the experimental objective and sample characteristics;

Optimize conditions: Optimize the concentration, incubation time, and temperature of the primary and secondary antibodies to obtain the best detection results;

Take precautions: DAB is a benzidine azo compound that can induce skin cancer and bladder disease. Take precautions during the color development process.

 

11.Counterstaining

 

Purpose: To form cell outlines and better localize the target protein, counterstaining can be performed.

Counterstaining: Add 80-100μL of hematoxylin staining solution to completely cover the tissue, incubate at room temperature for 5 minutes, differentiate with 1% hydrochloric acid alcohol for 1 second, rinse slightly with tap water, and then return to blue with tap water for 5 minutes.

Precautions:

The counterstaining time needs to be optimized, which is related to the preparation time of hematoxylin;

If the staining is too light, repeat the staining; if it is too dark, use hydrochloric acid for differentiation.

 

12.Mounting and Observation

 

Purpose: In immunohistochemical experiments, mounting and observation are the final steps used to protect the tissue sections, enhance contrast and stability, and allow for long-term observation and analysis under a microscope.

Mounting Steps:

1) Dehydration: After completing all staining steps, the tissue sections usually need to be dehydrated to remove moisture. The sections are sequentially immersed in 70%, 85%, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 1 minute each.

2) Clarification: After dehydration, the sections need to be clarified to allow the mounting medium to distribute evenly. Typically, the sections are immersed in xylene twice for 1 minute each.

3) Mounting: Add a drop of mounting medium to the tissue section, then gently cover it with a cover slip. The choice of mounting medium depends on the experimental requirements and sample type. When mounting, ensure that no bubbles are formed and that the cover slip is tightly attached to the section.

4) Drying: Allow the mounting medium to dry naturally, or accelerate the drying process in a warming box. The dried sections can be stored long-term and are ready for observation at any time.

 

It is hoped that this IHC experimental procedure guide will help you achieve satisfactory results in your experiments! If you have any questions or need further assistance, please feel free to contact Absin Biosciences at any time, and we will provide you with support.

 

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