Immunohistochemistry-Paraffin

Immunohistochemistry (IHC) is based on the principle of antigen-antibody specific reaction while proteins and other antigens in tissue sections are qualitatively and locatively studied. IHC can be divided into immunofluorescence method, immunoenzyme-labeled method and immunocolloidal gold technology according to the types of labeled substances. The main tissue samples used in the experiments include paraffin sections and frozen sections.

The basic principle of immunoenzyme-labeled method is to first interact with the tissue with enzyme-labeled antibody, and then add the enzyme substrate to generate colored insoluble products or particles with a certain electron density. Through light or electron microscopy, various antigen components on the cell surface and inside the cell are studied. At present, peroxide-antiperoxidase (PAP) method, vitelloin-biotin-peroxidase complex (ABC) method, Streptomyces biotin-peroxidase link (SP) method are widely used in pathological diagnosis.

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

1. Sampling

(1) Select the area to be observed and cut it into small pieces.
(2) Preliminary treatment: If the tissue sample contains blood or other body fluids, it should be rinsed with physiological saline or PBS to remove these substances that may affect the fixation effect.
(3) Fixation: Place the tissue sample in the fixative. Commonly used fixatives include 10% neutral buffered formalin (also known as formaldehyde), methanol, ethanol, or mixtures thereof. The amount of fixative is usually 10-20 times the tissue volume to ensure that the tissue is fully immersed in the fixative. Large tissue specimens should be cut and fixed to prevent autolysis and putrefaction of the middle part. The fixation time depends on the tissue type and experimental requirements, generally 18-24 h at room temperature or 4°C.
(4) After fixation, remove the tissue from the fixative and rinse it with running water for several minutes to remove residual fixative and any crystals that may form.

2. Embedding

(1) Dehydration: After the tissue sample is fixed, the water needs to be removed so that the embedding medium can fully penetrate it. Alcohol gradient dehydration: First, soak in 50% ethanol for 1-2 hours, then soak in 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol (I), and anhydrous ethanol (II) for 30-60 minutes each.
(2) Clearing: After dehydration, the tissue sample will become opaque. A clearing agent (such as xylene) is needed to remove the alcohol and other solvents, making the tissue sample transparent. Usually, soaking in xylene twice, 10-20 minutes each time, is used.
(3) Paraffin infiltration: Place the cleared tissue sample in molten paraffin, allowing the paraffin to fully penetrate the tissue sample. Generally, the paraffin is changed 3 times: the first time for 40 minutes, the second time for 1 hour, and the third time for 1 hour. This process usually needs to be carried out in an incubator, typically at 50-60°C, to ensure uniform paraffin penetration.
(4) Embedding: Place the paraffin-impregnated tissue sample into a mold, pour in molten paraffin, and allow the paraffin to cool and solidify. The tissue sample is then embedded in the paraffin.

3. Sectioning

(1) Ensure the embedded tissue block has cooled and solidified sufficiently. Use a suitable microtome to section.
(2) Sectioning: Fix the embedded tissue block onto the microtome's sample holder. Adjust the section thickness, typically 4-5 μM, depending on the antigen being detected and the experimental requirements.
(3) Spreading: Gently remove the sectioned tissue from the blade using a brush or paintbrush and spread it in warm water (40°C).
(4) Baking: Bake the section at 60°C for 2 hours. For heat-sensitive antigens, the section can be dried overnight at 37°C.

4. Dewaxing and Hydration

(1) Dewaxing: The slides are first immersed in xylene to dissolve and remove the paraffin wax. This process is typically performed in multiple steps, each lasting approximately 5 minutes, to ensure complete removal of the paraffin. For example, xylene I, xylene II, and xylene III can be used for 5 minutes each.
(2) Hydration: The slides are sequentially immersed in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and 70% ethanol, each for 5 minutes; then rinsed in purified water for 5 minutes.

5. Antigen Retrieval

Re-exposing or correcting these blocked or distorted antigens improves antibody-antigen binding rates in immunohistochemical experiments, enhancing signal strength and specificity.
The most commonly used retrieval buffers are 10 mM pH 6.0 sodium citrate, pH 9.0 Tris-EDTA, and pH 8.0 EDTA. It is recommended to test multiple methods to find the one with the best staining effect.
Antigen retrieval methods:
(1) Microwave retrieval: Add antigen retrieval solution, place the slide in the microwave, microwave on medium heat for 8 min, turn off for 7 min, then microwave on low heat for 8 min; remove the staining box and cool to room temperature.
(2) Water bath retrieval: Add antigen retrieval solution, place the slide in the microwave, and heat in a water bath. Continuously monitor the temperature with a thermometer until the antigen retrieval solution reaches an effective temperature (92℃). Maintain this temperature for 40 min, then remove the staining box and cool to room temperature.
(3) Autoclave retrieval: Add antigen retrieval solution to the staining box, place the slide in the microwave, and heat in an autoclave until it reaches full pressure. Continue heating for 5 min, turn off the power, and remove the staining box after 10 min. Cool to room temperature.
(4) Enzymatic hydrolysis and repair: Commonly used digestive enzymes include trypsin, pepsin, and proteinase K. Place the slides on a glass slide containing the hydrolysate, and then perform enzymatic hydrolysis under appropriate temperature and humidity conditions (usually 37°C, 20 min). After hydrolysis, the slides need to be cooled to room temperature before further processing.

6. Inactivation

Eliminate the influence of endogenous enzymes and active substances in cells or tissues, reduce background noise, and improve the specificity and sensitivity of the experiment.
(1) Inactivation of endogenous peroxidase: For the HRP detection system, the commonly used method to remove endogenous peroxidase is 3% hydrogen peroxide aqueous solution at room temperature for 10 min.
(2) Washing: Wash the slides with buffers such as PBS or TBS, and soak them twice, 5 min each time.

7. Blocking

Blocking non-specific binding sites on tissue sections prevents antibodies from binding to these sites, thereby reducing background signal.
(1) Non-specific site blocking: Add blocking solution (such as 5% BSA or serum) to the tissue section, ensuring that the blocking solution evenly covers the entire tissue area. Then place the section in a humidified chamber and incubate at room temperature or 37°C for 30-60 min to allow the blocking solution to fully react with the tissue section.
(2) Washing: After blocking, wash the section with buffer such as PBS or TBS to remove unbound blocking solution and any impurities that may be present.

8. Antibody incubation

Can be divided into direct methods (also known as one-step methods) and indirect methods (two-step, three-step, or multi-step methods). Direct detection methods are a one-step process in which the primary antibody directly binds to the conjugate (such as fluorescent dye, enzyme, colloidal gold, or biotin). The indirect method involves conjugating a secondary antibody to indirectly stain the tissue.
8.1 Direct Method
(1) Determine the optimal antibody dilution ratio to use. Dilute the antibody in TBST, adding 1% BSA as an additional blocking agent to reduce nonspecific binding.
(2) Incubate the tissue sections in the diluted primary antibody. Typically, incubate at room temperature for 1 hour or overnight at 4°C.
(3) Wash the tissue sections three times with PBST.
(4) Next, perform counterstaining, mounting, and imaging.

8.2 Indirect Method
(1) Determine the optimal antibody dilution ratio to use. Dilute the antibody in TBST, adding 1% BSA as an additional blocking agent to reduce nonspecific binding.
(2) Incubate the tissue sections in the diluted primary antibody. Typically, incubate at room temperature for 1 hour or overnight at 4°C.
(3) Wash tissue sections three times with PBST.
(4) Incubate the sample with pre-diluted secondary antibody. Incubation at room temperature for 30-60 minutes is generally recommended.
(5) Wash tissue sections three times with PBST.

9. Detection

Chemical staining or fluorescence staining can be used for staining.
9.1 Chemical Staining
(1) Immerse the slide in the chemical staining substrate solution.
(2) Wash the slide with tap water to remove excess staining solution.
(3) Wash the tissue sections with cold tap water to remove excess staining solution and make hematoxylin blue.
(4) Dehydrate the tissue using ethanol and xylene. Soak the sections sequentially in 75% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 3 min each. Then soak twice in xylene for 5 min each time.
(5) Add a few drops of mounting medium to the slide: Let the slide stand at room temperature for 5 minutes.
(6) Carefully place the coverslip onto the slide using tweezers.
(7) Image the tissue section using a microscope. If not used immediately, store the slide at 4°C.

9.2 Fluorescence Developing Method
(1) Immerse the tissue section in counterstaining solution (optional).
(2) Wash the slide with ice-cold deionized water to remove excess staining solution.
(3) Add a few drops of mounting medium suitable for fluorescence developing method to the slide. Let stand at room temperature for about 5 minutes.
(4) Carefully place the coverslip onto the slide using tweezers.
(5) Image the tissue section using a fluorescence microscope. If not used immediately, store the slide at 4°C in the dark.

1. The dewaxing process must be thorough to ensure complete removal of paraffin. Otherwise, residual paraffin will affect subsequent antigen detection and staining.
2. Throughout the dewaxing and hydration process, the slides should be kept moist to avoid drying. Drying can lead to nonspecific antibody binding, resulting in high background staining.
3. Allow the slides to cool naturally after heating; use an excess of antigen retrieval buffer.
4. Different pH values ​​of the retrieval buffer have a significant impact on staining results.
5. 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 must be selected based on experimental requirements and antibody characteristics.
6. If there are concerns about the presence of endogenous biotin or peroxidase in the sample, endogenous avidin/biotin blocking and endogenous peroxidase blocking can be performed.
7. When incubating secondary antibodies in the indirect method, if a fluorophore is used, incubation must be performed in the dark.
8. During antibody incubation, the antibody solution must completely cover the sample.
9. If the mounting medium you are using contains a fluorescent counterstain, an additional counterstaining step is not required.
10. Mounted sections can be stored for a long time, but should be placed in a dry, dark environment at a suitable temperature to prevent the sections from becoming damp, fading, or damaged.