Everything About Immunohistochemistry (IHC) Principle
Immunohistochemistry (IHC) is a technique used to find specific proteins (called antigens) in cells within a tissue sample. It works by using special proteins called antibodies, which are designed to stick only to the target antigen.
To see where the antibody has attached, a chemical reaction is used to create a visible color. This is typically achieved with enzymes such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), which facilitate the color change.
In many cases, a secondary antibody DAB staining method is used—where a secondary antibody (linked to HRP) binds to the primary antibody, and reacts with DAB (3,3'-diaminobenzidine) to produce a brown color at the site of the antigen.
IHC is commonly used in both research and medical labs because it shows exactly where certain proteins are located inside cells and tissues. There are different ways to perform IHC, and the best method depends on factors such as the type of tissue being tested and the level of sensitivity required.
Immunostaining Methods: Direct, Indirect, and Amplification
Direct Method
This method uses primary antibodies that are already labeled with a fluorescent dye or enzyme.
Since the label is already on the antibody, the process is quicker.
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Pros: |
Cons: |
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Great for doing multiple stains, especially with fluorescent dyes. |
You need a lot of labeled antibodies, which can be expensive. |
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No background staining from secondary antibodies. |
Not many labeled primary antibodies are sold. |
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Faster process. |
Sometimes, labeling the antibody can reduce its ability to bind properly. |
Indirect Method
Uses unlabeled primary antibodies, followed by labeled secondary antibodies—such as those provided by AAA Biotech—that bind to the primary ones
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Pros: |
Cons: |
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Flexible: You can use the same secondary antibody for many different primary antibodies (as long as they come from the same host species). |
Harder to stain multiple targets at once, because primary antibodies must come from different species. |
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Takes more time because of the extra step. |
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Secondary antibodies might cause non-specific background staining. |
Amplification Method
Uses biotin-labeled primary antibodies and a special molecule (avidin or streptavidin) that binds to biotin and is also labeled.
Some companies also make special secondary antibodies that boost signal strength.
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Pros: |
Cons: |
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Very helpful when the target molecule is in low amounts, because it amplifies the signal. |
You have to be careful if the tissue has natural biotin, as it can give false signals. |
Types of Tissue Sections
Paraffin Sections (FFPE)
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Tissues are fixed with a chemical like formalin, then embedded in wax (paraffin) and sliced into thin sections.
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These samples are great for long-term storage and are widely used.
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But: the preparation takes time, and the structure of the proteins might change during processing.
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Because of this, antigen retrieval (a step to uncover the target protein) is often needed.
Frozen Sections
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Tissues are frozen and sliced without using chemicals or wax.
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Used mainly in surgeries for quick diagnosis.
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They preserve protein structure and activity, so no antigen retrieval is needed.
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But: they don’t store well long-term.
Staining Procedure for FFPE Tissue (Indirect Method)
(Example based on MBL protocol)
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Bake slides – Heat at 60°C for a few hours to soften the wax.
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Remove paraffin – Use chemicals like xylene and ethanol.
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Wash – Clean off any remaining chemicals.
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Antigen retrieval – Uncover hidden proteins to help antibodies bind.
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Wash – Rinse again.
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Block natural peroxidase – Stop the tissue’s own enzymes from reacting.
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Wash
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Blocking step – Prevent non-specific antibody binding.
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Add primary antibody – Leave at room temperature for 1–2 hours.
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Wash
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Add labeled secondary antibody – Leave at room temperature for 1 hour.
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Wash
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Counterstain – Add a dye (e.g., hematoxylin) to stain cell parts like nuclei.
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Dehydrate and clear – Use ethanol and xylene to remove water.
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Mount the sample – Seal with a mounting medium for storage.
Why Each Step Matters?
Baking & Deparaffinization
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Paraffin blocks water and antibodies, so it must be removed.
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Baking melts the paraffin; xylene and ethanol wash it away.
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Xylene is toxic, but safer options exist.
Antigen Retrieval
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Fixing tissue can hide target proteins (antigens).
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This step uncovers them so antibodies can bind.
Two main methods:
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Heat (e.g., in citrate or Tris-EDTA buffer)
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Enzymes (e.g., proteinase K, pepsin)
You must adjust the method, pH, temperature, and time depending on the antibody.
Inactivating Natural Peroxidase
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Tissues naturally contain peroxidase, which can react and cause false staining.
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Treating with 3% hydrogen peroxide removes this.
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If skipped or done incorrectly, it may lead to background staining.
Counterstaining
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Adds contrast to make tissue structures easier to see.
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Hematoxylin stains nuclei blue or purple.
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Color may change depending on water and temperature, so be careful.
Dehydration, Clearing & Mounting
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If you're using a non-water-based (hydrophobic) mounting medium:
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First, remove all water using ethanol.
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Then use xylene to clear the tissue.
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This step isn't needed for water-based mounting media.
Mounting
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Seals and protects the slide for long-term use.
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Hydrophobic (non-water-based) media like xylene-based ones are best for storage.
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Prevents fading and drying of the stained sample.
