At HistoSpring, we are all about clarity. Clarity of slides, stains, protocols and, most importantly, communication. To provide our research partners with the tools and information needed to succeed, we had expert, Sallie Schneider, Ph.D., Director of Histology to answer some of the most commonly asked questions we encounter in our histology lab.
#1) I am not seeing any staining with my antibodies. What is the problem?
It’s frustrating when you don’t see staining with your antibodies, and it’s a very common issue. A study estimated that roughly US $28.2B is spent on irreproducible preclinical research in the United States, of which roughly 36% is attributed to problems with reagents, particularly antibodies. That’s a significant figure, and it highlights the importance of getting this right.
So, when you encounter a lack of staining, it’s essential to approach the issue systematically. First, let’s examine the antibody itself. Was it a commercially acquired antibody, and has it been validated specifically for immunohistochemistry (IHC) and your species of interest? Optimization is a key factor. Have you explored varying antibody concentrations, antigen retrieval methods, and incubation times to refine your protocol? Additionally, ensure that your secondary antibody is compatible with your primary.
It’s also important to consider the impact of fixation. Certain epitopes are sensitive to prolonged exposure to fixatives. Transferring tissues to 70% ethanol within 24 hours can mitigate this issue.
Another consideration is how the source of your antibody can influence results. In-house or “homegrown” antibodies, while sometimes effective, can present unique challenges compared to rigorously validated commercial antibodies. It’s worth noting that an antibody may perform well in immunocytochemistry (ICC) but not in IHC. Antibody source considerations along with any issues in storage, dilution, and species compatibility are some of the most likely culprits of absent staining. Always make sure your positive control is functioning correctly to validate your antibody and procedure.
#2) How do you select control tissues for IHC assays?
The selection of appropriate control tissues is fundamental to ensuring the accuracy and reliability of immunohistochemistry (IHC) assays. Positive control tissues, demonstrating expression of the target antigen, serve as a critical benchmark for successful staining. Conversely, negative control tissues, which lack the target antigen, are essential for identifying non-specific binding and background staining. The concurrent use of both positive and negative controls is imperative for verifying the assay’s specificity to the intended target antigen and confirming that any observed staining results from specific antibody-antigen interactions.
#3) I am getting staining in areas/cells that I didn’t expect. What is the problem?
If staining is observed in unintended regions, ask yourself the following questions:
- Has your antibody concentration been thoroughly optimized? It’s possible the concentration is too high.
- Are you utilizing an appropriate blocking agent, such as serum, BSA, or a commercial blocking solution?
- Have you considered the species of your antibodies and tissue? If you are using mouse antibodies on mouse tissue, endogenous immunoglobulins may be contributing to background staining.
- Is your tissue mucinous tissue? Non-specific antibody binding could be a contributing factor.
- For DAB detection, have you employed hydrogen peroxide (H₂O₂) to block endogenous peroxidase activity?
Lastly, if you lack a robust negative control, a practical approach is to stain tissues without the primary antibody. This will help determine if your secondary antibody contributes to the non-specific staining.
#4) Can I stain with several different antibodies on the same tissue section?
The short answer is yes, though careful consideration of potential cross-reactivity is crucial. If each primary antibody is directly linked to a distinct fluorophore, cross-reactivity is typically not a concern. However, when relying on secondary antibodies, it is imperative to ensure that each secondary antibody binds exclusively to its intended primary antibody to prevent cross-reactivity. It is also worth noting that several new multiplex kits have been introduced in recent years, which greatly simplify these types of assays.
#5) We’ve had to reduce our lab staff significantly due to budget constraints. Are you able to provide staining services for us?
These are definitely uncertain times, and funding can fluctuate throughout grant cycles. HistoSpring is here to support you whether you require tissue processing, embedding, and/or sectioning. If your staffing constraints prevent you from performing staining in-house, we can also handle the staining, or we can tailor our services precisely to meet your lab’s unique requirements.
At HistoSpring, our team of experts provides active collaboration and tactical solutions to ensure successful results. Our deep understanding of chemistry, diverse staining techniques, and proficiency in troubleshooting staining issues for tissue sections and analyzing multiple biomarkers sets us apart. We offer a comprehensive range of services, from commonly used stains to highly specialized and custom staining protocols, all leveraging state-of-the-art automated histology to improve research and clinical outcomes.
HistoSpring acts as a reliable partner for your research—proactively identifying and resolving potential issues, ensuring your projects stay on track, and delivering the highest quality results, even in challenging circumstances. Consider us an extension of your team, ready to adapt and provide optimal solutions.
