IHC Staining


Immunohistochemistry (IHC) uses antibodies to visualize protein localization within tissue sections. Protein expression patterns are essential for disease diagnosis. A successful IHC outcome relies on optimized staining protocols and high-quality research tools.

Tissue processing – fixation and sectioning.

Tissue fixation and embedding are essential steps in preserving antigens, maintaining tissue structure, and preserving protein antigens. Scientists can select between paraffin embedding, frozen sections, and floating sections depending on the specific needs of the IHC study (Table 1).

Table 1 IHC - paraffin, freezing and floating.
Paraffin-embedded tissue Frozen tissue Floating Sections


Incubate with formaldehyde overnight or 24 hours.

Use formaldehyde, methanol, ethanol, or acetone.

Use formaldehyde.


Dehydrate tissue and embed in pre-heated paraffin (65ºC) then cool to allow the paraffin to harden.

Immerse tissue in liquid nitrogen or isopentane.

Not applicable.






Years at room temperature.

2 years at -190°C.

Around 1 to 2 weeks at 4°C.


Allows for long-term storage.

Shorter protocol compared to paraffin-embedded tissues. Useful for fast diagnosis during surgery.

Thick sections allow for visualizing 3D structure of the tissue.


Fixation masks the antigen – may require antigen retrieval step.
Protocol is more time consuming due to gradual rehydration prior to, and dehydration after the IHC experiment.

Crystals forming during freezing step, thus impacting tissue structure.
Requires endogenous enzyme activity to be inactivated.

Difficult to visualise individual cells due to thickness of the tissue.

Antigen Retrieval

Formaldehyde fixation may mask epitopes and prevent antibody binding. Antigen retrieval may be required to reveal the epitopes required for antibody binding. There are two main methods – table 2.

Table 2 Epitope Retrieval
Heat-induced epitope retrieval Proteolytic-induced epitope retrieval


Approximately 95°C.



Usually pH 6 (higher pHs if needed).

pH 7.4.

Incubation time

10–20 minutes.

10–15 minutes.

Buffer composition

Popular buffers include sodium citrate, EDTA, and Tris-EDTA.

Pepsin, proteinase K or trypsin.

Blocking proteins

To prevent non-specific antibody binding to tissue or Fc receptors, it is crucial to block with serum or a protein blocking reagent. A serum from the same species as the secondary antibody is an ideal blocking agent. Additionally, proteins like bovine serum albumin (BSA) or casein can effectively block non-specific antibody interactions.

Please see below our blocking reagents:


In avidin/biotin-based detection systems, endogenous biotin can lead to false positives due to its widespread presence in tissues like the kidney, liver, and brain. To address this, blocking endogenous biotin is recommended. This involves blocking before avidin incubation and then incubating with biotin to prevent additional biotin binding to avidin. Products which may be of interest include:

Blocking Endogenous Enzymes

In enzymatic detection methods, it is essential to block endogenous enzymes to avoid non-specific staining. This is particularly important for chromogenic detection methods, which rely on the activity of enzymes conjugated to antibodies to visualize their localization.

Treatments like hydrogen peroxide (H2O2), which are commonly used to block endogenous enzymes, can damage epitopes and affect antibody binding. Therefore, it is generally recommended to block endogenous enzymes after incubating with the primary antibody to minimize this risk.

However, if the antibody is an HRP primary conjugate, the blocking step needs to be done prior to the addition of the primary conjugate.

Peroxidase Blocking

Endogenous peroxidase activity is a common source of non-specific staining in HRP-based detection methods. To check for endogenous peroxidase activity, incubate tissues with DAB substrate before primary antibody incubation. The tissues turning brown signifies the presence of endogenous peroxidase which will require blocking. Hydrogen peroxide (H2O2) is the most common peroxidase blocking agent which works by inactivating peroxidase enzymes. Relevant products include:

Alkaline Phosphatase Blocking

Endogenous alkaline phosphatase (AP) activity can also cause non-specific staining in AP-based detection methods. To test for endogenous AP activity, incubate tissues with BCIP/NBT. If the tissues turn blue, then endogenous AP is present and needs to be blocked.

Common AP inhibitors include Levamisole hydrochloride and tetramisole hydrochloride. Please see:

Primary Antibody

Selecting the correct antibody is key for a successful IHC experiment. Key considerations include:

  1. Does the antibody react bind to my target species? For instance, an antibody against a human protein may not detect the mouse equivalent if the antigen is not conserved.
  2. Is the antibody specific against the protein – usually demonstrated through using knockout-cell lines in western blot (usually a single band in the wild type control) or ICC experiments.
  3. Signal to noise ratio.
  4. Does the antibody demonstrate consistent staining patterns when using positive and negative tissues (The Human Protein Atlas is a rich resource containing IHC results for a large number of antibodies)
  5. Clonality – customers may use polyclonals as well as monoclonals. If using polyclonals, it is essential to ask the manufacturer if they batch test in IHC to ensure batch to batch consistency.
  6. Direct vs Indirect detection.
Direct detection Indirect detection

Used to detect highly expressed proteins.

Protocol may be used with low as well as high expressing antigens.

Protocol does not use secondary antibodies.

Signal is amplified and therefore useful for detecting lowly expressed proteins.

Useful for multicolor experiments.

Detection - Chromogenic vs Fluorescent Detection

Fluorescent staining

  • Multiplexing capabilities: Fluorescent staining allows for the simultaneous visualization of multiple antigens using different fluorophores excited at distinct wavelengths, enabling a more comprehensive analysis of protein interactions and expression patterns.
  • Superior image quality: Fluorescent methods generally produce higher-resolution and more detailed images compared to peroxidase-based staining, allowing for more precise localization and characterization of protein targets.
  • Signal amplification: Fluorescent signals can be amplified using HRP antibody labels coupled with tyramide-dyes, resulting in enhanced sensitivity for detecting low-abundance proteins.
  • Quantifiable results: Fluorescent staining is generally more amenable to signal quantification, enabling the measurement of protein expression levels with greater accuracy.
  • Autofluorescence limitations: Formaldehyde fixation, commonly used in immunohistochemistry, can induce autofluorescence in certain tissues, potentially interfering with fluorescent signal detection.
  • Efficient protocol: Fluorescent staining procedures are generally faster and less labour-intensive compared to peroxidase-based methods, reducing overall experimental time.
  • Cost considerations: Fluorescent imaging equipment is typically more expensive than standard brightfield microscopes, which may be a factor in some research settings.
  • Fluorophore stability: Fluorophores can exhibit fading over time, particularly upon exposure to light or harsh environmental conditions, potentially affecting long-term storage of fluorescently stained samples.


Chromogenic staining

  • High signal amplification: Biotinylated secondary antibodies and streptavidin-HRP can further amplify the signal in an ABC method, allowing for detection of low-abundance proteins. Alternatively, you can use a modern HRP-polymer secondary antibody, which offers similar amplification without the need for multiple incubation steps.
  • Long-term storage: Some precipitates, such as HRP/DAB, are photostable and can be stored for many years without fading. This makes peroxidase-based methods suitable for applications where long-term storage is important.
  • Standard equipment: Only a standard brightfield microscope is required for visualization of peroxidase-based staining.
  • Wide-area staining: The enzyme/chromogen precipitate is deposited over a wider area than photons from a fluorescent source, which can provide a more comprehensive view of protein expression.
  • Potential for longer procedure: The procedure for peroxidase-based staining generally includes more incubation and blocking steps than fluorescent methods, which can prolong the overall protocol. However, the exact duration depends on the chosen amplification system.
  • Challenges in quantification: Quantification of protein expression can be more difficult with peroxidase-based methods due to enzymatic amplification, which can introduce variability in signal intensity.
Table 3 Popular chromogens for enzymes.
Enzyme Chromogen / Substrate Color Mounting Media Advantages




Organic / aqueous

Intense color; permanent

DAB + nickel enhancer


Organic / aqueous

Intense color; permanent





Intense color; contrasts well with blue in double staining


Blue / Black


Intense color



Permanent Red


Organic / aqueous

Multi-color IHC

Fluorescent IHC allows for the analysis of multiple proteins on the same tissue section. Using primary conjugated antibodies, instead of a primary plus secondary conjugated antibodies, simplifies the protocol and allows primary antibodies raised in the same species to be used simultaneously.

Multi-color IHC allows for the generation of a large data set from a single tissue slide, thus reducing the amount of tissue required.


Useful for staining cellular structures within the tissue and therefore allows visualisation of antibody staining using the cellular structure as a reference point. The most common counterstain is hematoxylin which colors the nuclei blue contrasting with the brown HRP-DAB.

Table 4 Counterstains and their targets
Type Dye Target Color



Blue to violet


Nucleic acids



Nucleic acids