Spotting signs of disease often starts with noticing small changes—like how cells or tissues look under a microscope, or shifts in biomolecule levels within a sample. Immunohistochemistry (IHC) is a key method scientists use to highlight those changes. By applying targeted antibodies that bind to specific proteins, IHC allows researchers to visualize cellular features linked to disease, treatment response, or overall health.
Because IHC relies on a series of carefully timed and controlled steps, consistency is essential. That’s why many labs turn to IHC autostainers—automated instruments that handle the staining process with speed and precision. These systems help improve reproducibility, reduce manual effort, and support more confident interpretation of results.
In this guide, we’ll break down how IHC autostainers work, what to consider when choosing one, and how different features can support your research or diagnostic goals.
What is IHC?
Immunohistochemistry, or IHC, is a method used to detect specific biomolecules in cells and tissues using antibodies. These antibodies bind to their targets with high specificity, allowing scientists to visualize the presence, location, and abundance of key proteins under a microscope.
The technique combines the precision of immunology with the spatial detail of histology—offering a powerful way to study disease markers, cell signaling, and treatment effects at the tissue level.
IHC relies on the unique structure of antibodies:
- Core (constant) region: Acts as the structural backbone.
- Variable region: Binds to a specific antigen through chemical interactions like hydrogen bonding and electrostatic attraction.
dOnce bound, the antibody is detected through either a color-producing chemical reaction or a fluorescent signal—depending on the staining method used. We'll explore those next.
IHC techniques: CIH vs. IF
There are two main methods for visualizing antibody binding in IHC: chromogenic immunohistochemistry (CIH) and immunofluorescence (IF). Both approaches produce a detectable signal under a microscope, but they differ in how that signal is generated and interpreted.
Chromogenic IHC (CIH)
CIH uses enzymes attached to the antibody to produce a visible color change when exposed to a chemical substrate.
- Enzymes used: Typically horseradish peroxidase (HRP) or alkaline phosphatase (AP)
- Signal type: Colored precipitate (often blue or brown)
- Detection: Viewed under a standard light microscope
This method is widely used in clinical diagnostics due to its simplicity and compatibility with archived samples. It produces durable, easy-to-interpret stains that can be reviewed over time.
Immunofluorescence (IF)
In IF, antibodies are tagged with fluorescent dyes rather than enzymes. When excited by a light source, these dyes emit light at specific wavelengths, creating a fluorescent signal.
- Signal type: Fluorescent emission
- Detection: Requires a fluorescence microscope
- Advantages: Higher sensitivity, better signal-to-noise ratio, and the ability to detect multiple targets in the same sample (multiplexing)
IF is commonly used in research settings, especially when detailed spatial or quantitative information is needed.
Common clinical and research applications
IHC plays a central role in both diagnostics and research. Its ability to reveal molecular features within intact tissue makes it an essential tool for identifying disease, characterizing cell populations, and studying treatment responses.
Clinical applications
IHC has been used in clinical diagnostics for nearly a century, particularly in oncology and infectious disease pathology. It helps clinicians detect and classify diseases based on molecular markers present in tissue samples.
Examples include:
- Tumor identification: IHC can reveal where a tumor originated and how it’s behaving. For example, staining for smooth muscle actin helps distinguish muscle tumors from other gastrointestinal tumors.
- Lung cancer diagnosis: Different subtypes of lung cancer express different markers. Adenocarcinomas often test positive for TTF1, while squamous cell carcinomas express p40. IHC helps pathologists make these distinctions microscopically.
- Infectious disease detection:
- Syphilis: IHC can detect Treponema pallidum using antibodies that target unique microbial proteins.
- Bacteremia: Researchers have used IHC to identify Bartonella henselae in tissue samples from individuals exposed to cat scratches, even those with healthy immune systems.
Research applications
In research labs, IHC is used to study disease mechanisms, identify potential biomarkers, and evaluate experimental therapies. Its ability to preserve tissue structure while highlighting molecular details makes it ideal for exploratory and translational studies.
IHC workflow: 4 essential steps
Producing high-quality, reproducible IHC results depends on following a consistent and well-optimized workflow. While details may vary by protocol or sample type, most IHC assays follow the same four core steps:
1. Fixation
Fixation preserves the structure of cells and tissues, preventing degradation and maintaining antigen integrity.
- Formalin (formaldehyde in water) is the most common fixative. It works by cross-linking proteins to stabilize cell architecture. Formalin-fixed tissues are usually embedded in paraffin for sectioning. However, over-fixation can mask target proteins, reducing antibody access.
- Alcohols like methanol and ethanol are also used. They don’t mask antigens, but they can lead to protein degradation, which may affect antibody binding.
2. Antigen retrieval
This step is usually needed for formalin-fixed, paraffin-embedded samples. Cross-linked proteins may block antibody binding, so heating the tissue (often in a buffer solution) helps “unmask” the antigens and improve staining.
3. Blocking
To reduce background signal, a blocking step prevents antibodies from sticking to non-target components.
- Blocking agents may include proteins, avidin/biotin solutions, or hydrogen peroxide, depending on the detection system.
- This step is especially important for minimizing noise in complex tissue samples.
4. Antibody labeling and visualization
After preparation, tissue sections are ready for staining:
- Primary antibody binds to your target molecule.
- Secondary antibody (optional) binds to the primary antibody to amplify the signal.
Whether or not to use a secondary antibody depends on your experimental needs:
- Use secondary antibodies if you need a stronger signal—especially when detecting low-abundance targets.
- Skip them if specificity is a concern, since secondary antibodies can sometimes bind non-specifically and introduce background staining.
If you’re interested in learning more about different detection and signal-amplification methods—such as polymer-based systems or biotin‑streptavidin techniques—check out this detailed guide from Abcam on detection and amplification systems in IHC.
When choosing between strategies, ask yourself:
- Do I need stronger signal? Amplification methods (like using secondary antibodies, polymer systems, or biotin–streptavidin) can boost sensitivity—especially useful for low-abundance targets.
- How important is specificity? More complex amplification may increase background noise, so weigh this when precision is critical.
Why autostainers matter
Running an IHC assay manually requires careful timing, consistent technique, and attention to every detail—from fixation to antibody incubation. Even small variations can impact staining quality and reproducibility.
However, a 2021 study found that automating the IHC workflow significantly improved staining uniformity and consistency compared to manual methods, especially in large or complex tissue sections.
Autostainers take the guesswork out of this process by automating each step in the IHC workflow. With the right system in place, labs can:
- Reduce hands-on time and technician variability
- Improve consistency from slide to slide and batch to batch
- Scale up throughput without sacrificing quality
- Standardize staining across different users, sites, or time points
For clinical labs, this means more reliable diagnostic results. For research teams, it means stronger, more reproducible data. Whether you're staining 10 slides or 100, automation helps ensure your IHC workflow runs smoothly and predictably.
Comparing top IHC autostainers
A number of commercial autostainers are available, each with different capacities, features, and workflow integrations. One important consideration is reagent efficiency—how much staining solution the system uses and whether it minimizes waste.
According to a study, enclosed staining chambers with controlled reagent flow significantly reduce both the volume of reagents needed and background staining levels compared to open-slide systems.
Here’s a side-by-side look at some widely used systems:
Each system balances speed, flexibility, and automation differently. Some prioritize throughput, while others emphasize staining precision, reagent efficiency, or software integration.
Choosing the right autostainer for your lab
Finding the right IHC autostainer isn’t just about features—it’s about how weAccording to a study, enclosed staining chambers with controlled reagent flow significantly reduce both the volume of reagents needed and background staining levels compared to open-slide systemsll the system fits your specific needs. Here are a few important questions to ask as you evaluate your options:
- What kinds of samples are you working with? Different tissue types and sample conditions can affect staining outcomes. Some autostainers offer more flexibility for custom protocols, while others are optimized for standard clinical specimens.
- How many slides do you need to process per day? Throughput matters—especially if you’re running multiple assays or supporting multiple investigators. Make sure the system’s capacity and run time match your lab’s daily or weekly volume.
- Are you working with established or novel biomarkers? If you’re using validated antibodies and protocols, systems with built-in assay menus can save time. But if you’re developing new markers, you may need a platform that supports custom staining protocols and greater experimental flexibility.
- Do you want control over consumables? Some systems require the use of proprietary reagents, which can streamline validation but limit flexibility. Others are more open, allowing you to choose your own antibodies and detection reagents.
Taking time to think through these questions will help ensure you choose a system that supports your scientific goals, budget, and workflow—now and as your lab evolves.
Let’s talk about your IHC setup
Reproducibility is essential in IHC work. Automating your staining protocol with an autostainer can help you generate consistent, high-quality results—making it easier to detect disease markers, track progression, and evaluate treatment effects with confidence.
Whether you’re processing a few slides or hundreds each day, choosing the right autostainer can make a meaningful difference in your workflow. We work with a wide range of brands and models, so you can find the system that best fits your lab’s goals—without being locked into a specific inventory.
Use this guide as a starting point, and when you’re ready, get in touch. We’re here to help you explore your options, answer questions, and figure out whether leasing an IHC autostainer is the right move for your lab.