Selecting Antibodies For IHC
Tips to help you select the right antibody for your IHC staining.
High-quality antibodies are desirable for their high specificity and low cross-reactivity, generating reliable and successful staining. When considering a primary antibody for IHC, there are certain factors you should consider:
- Number of citations
- Consult literature and antibody comparison resources
- View the in-house validation data from the antibody manufacturer to ensure that the antibody is validated in the correct application in native samples
Selecting a Primary Antibody for IHC
When choosing a primary antibody, the question of clonality always comes up. While polyclonal antibodies are used more often in IHC, the decision as to whether to use a polyclonal or monoclonal antibody depends on the target of interest and sample type.
Advantages and Disadvantages of Polyclonal and Monoclonal Antibodies in IHC
Advantages of Polyclonal Antibodies in IHC | Advantages of Monoclonal Antibodies in IHC |
---|---|
Heterogeneous population | Homogeneous population |
Recognizes multiple epitopes | Specific for a single epitope |
Less sensitive to changes in pH, tissue, buffer, protein conformation | Can detect a single protein with high affinity, even if it shares sequence similarities with other proteins |
More likely to cross-react in rarer species | Good for long-term projects, minimal lot-to-lot variability |
Disadvantages of Polyclonal Antibodies in IHC | Disadvantages of Monoclonal Antibodies in IHC |
---|---|
Not specific for one epitope, can lead to cross-reactivity with similar proteins | Sensitive to changes in pH, tissue, buffer, protein conformation |
Possibility of lot-to-lot variability | Lower avidity can be an issue for low expressed targets |
Less chance of cross-reactivity with rarer species |
Optimizing a Primary Antibody for IHC
The optimal conditions for a primary antibody may vary between experiments and tissue type. Below are some general steps on how to optimize your primary antibody for each IHC experiment:
- Try different antibody dilutions based on manufacturer guidelines. 1:50 or 1:300 are good starting points for optimization, but you may have to go lower to get the best staining.
- If you have specific staining, but high background signal, try varying incubation time and temperature. In this case, shorter incubations at room temperature are recommended.
- If your antibody is high-affinity and at a high concentration, try incubating for a shorter time.
- If your antibody is high affinity but at a low concentration, try increasing the incubation time and lowering the incubation temperature.
Selecting and Optimizing Secondary Antibodies for IHC
Selecting the right secondary antibody for your needs is essential to help reduce signal-to-noise ratio, leading to better quality staining. Below are some points to be aware of when choosing a secondary antibody:
- Subclass specificity: Polyclonal primaries are mainly IgG isotypes. Monoclonal primaries are occasionally of different isotypes and hence require an isotype-specific secondary antibody. For example, mouse-derived antibodies have four IgG subclasses: IgG1, IgG2a, IgG2b, and IgG3. To select an antibody that targets all subclasses, make sure that it is against the “heavy and light” (H+L) chain.
- Cross-adsorption: Secondary antibodies can go through an additional purification step to reduce potential cross-reactions with other species. Here, the secondary antibody solution is passed through different columns containing sera of different species to filter out the non-specific antibody.
- F(ab’)2 fragments: High background staining can be due to the presence of Fc receptors in certain tissue or cells (lymph nodes, spleen, macrophages, etc.). A whole antibody can bind to the Fc region, while an F(ab’)2 fragment does not, ensuring higher specificity. These fragments also allow for better tissue penetration due to their smaller size.
Multi-rAb Recombinant Secondary Antibodies
Multi-rAb Recombinant Secondary Antibodies are finely crafted mixtures of recombinant goat monoclonal antibodies that recognize multiple complementary epitopes on the same IgG. Each recombinant clone in the final multiclonal mixture is carefully selected after rigorous characterization and screening to ensure high specificity, minimal cross-reactivity, and low background. Since Multi-rAbs are generated using a recombinant platform, they also ensure high lot-to-lot consistency resulting in highly reproducible results.
For chromogenic IHC, our Multi-rAb Polymer HRP Recombinant Secondary Antibodies enable highly sensitive, biotin-free detection of mouse or rabbit primary antibodies. Unlike traditional avidin-biotin-based signal amplification methods, polymer HRP-based detection offers advantages of lower background and faster protocol with fewer steps. Therefore, we recommend using our Multi-rAb polymer HRP secondary antibodies for chromogenic staining workflows.
We also offer Multi-rAb Recombinant Secondaries conjugated to various CoraLite® Plus dyes for fluorescent IHC staining.
IHC Controls
To ensure that the observed staining pattern is specific and not due to any cross-reactivity or non-specific binding, it is important to include control slides as part of your experimental design. Controls should be run alongside the main experiment.
Reagent Controls
- Secondary Only Controls
- Process the slide as normal but omit the primary antibody.
- This ensures the signal detected is specific for the target.
- If multiplexing, test all fluorophores in isolation.
- Endogenous Only Control
- Process the slide as normal but omit the primary and secondary antibody.
- This reveals the level of autofluorescence/artifacts.
- Isotype Control
- Replace the primary antibody with a non-immune IgG of the same species, isotype, and concentration as the primary antibody.
- Ensures observed signal is specific for FAB paratope-epitope binding.
Antigen Controls
- Positive Control
- Control tissue that is known to express the protein of interest.
- Confirms if the protocol is correct.
- Validates negative results.
- Negative Control
- Control tissue that is not known to express the protein of interest.
- Ensures that the observed staining pattern is due to specific signals.
- Use www.uniprot.org or www.pax-db.org to find endogenous negative controls in tissues.
- CRISPR knockouts or siRNA knockdown cell lines are good negative controls.
- Checking mRNA levels of target proteins can also serve as a good negative control.