Creating a product that stands out and addresses the needs of scientists requires ingenuity, persistence, and a lot of testing; our new product, VectaPlex™ Antibody Removal Kit, is no exception. We sat down with Senior Scientist August Estabrook, PhD, to get an inside look into the birth and development of VectaPlex. 

What is the VectaPlex Antibody Removal Kit? 

VectaPlex is not just another reagent kit. It’s a carefully crafted ensemble of reagents designed specifically to strip detection reagents from tissue specimens without damaging them. Its magic lies in allowing scientists to perform sequential staining of multiple markers, even when using antibodies from the same species, thereby achieving a rich, multiplexed image that can reveal intricate details about the sample. 

How does VectaPlex stand out from current antibody removal methodologies? 

Today, many labs rely on microwave treatment or pressure cooking when it comes to stripping detection reagents. But these methods, despite their widespread use, have a downside: they can be aggressive, often compromising the delicate morphology of the tissue samples. VectaPlex, on the other hand, offers a gentler approach. It effectively removes the antibodies without causing harm to the tissue, establishing it as a unique and superior solution in the realm of antibody removal. 

Where did the idea of VectaPlex come from? 

In our continuous pursuit of advancing research, we identified a pressing need: the power of multiplexing was clear, but there wasn’t an easy way to use primary antibodies from the same species without risking cross-reactivity. The idea for VectaPlex was born out of this very need, aiming to address this gap and provide researchers with a tool that simplifies complex procedures. 

How did VectaPlex go from an idea to a finished product? 

The journey from concept to product is never linear. Armed with an idea, we delved deep into the existing literature, gleaning insights and identifying gaps. Starting with preliminary formulations, we embarked on a process of refinement. It involved multiple iterations, each time inching closer to our goal.   

What were the major challenges during the development of VectaPlex? 

Every innovation comes with its set of challenges. For me, aligning the images using a computer program was a steep learning curve. But innovation thrives in collaboration. With the support and guidance from colleagues who were experts in the area, what seemed like an insurmountable challenge became a manageable task.  

What made the development process of VectaPlex rewarding? 

There’s an indescribable joy in seeing one’s efforts come to fruition. For us, that moment arrived when we achieved our first 6-Plex image using solely mouse antibodies. Witnessing that vibrant, detailed image was not just a testament to VectaPlex’s capabilities but also a heartwarming validation of our hard work. 

How will VectaPlex benefit its users? 

VectaPlex is more than a product; it’s an enabler. It lowers the hurdles researchers often face in achieving multiplexing. By using VectaPlex, researchers no longer have to grapple with the complexities of specialized equipment or invest in expensive labeled primaries. It democratizes the process, allowing everyone to embrace the power of multiplexing with their standard reagents and workflows. 

Any insights or tips for potential users of VectaPlex? 

Diving into a new method can seem daunting. However, we’ve got you covered. Our comprehensive user guide breaks down the process, making it straightforward and easy to follow. With VectaPlex by your side, multiplexing with your regular reagents becomes a seamless experience. 
 

For those looking to push the boundaries of their research and delve deep into the world of multiplexing, our Immunofluorescence Resources Guide and Multiplexing Guide are invaluable resources. Stay tuned to our blog for more insights, innovations, and behind-the-scenes stories. 

Multiplexing, a powerful technique that allows the visualization of multiple biomarkers within a single sample, has revolutionized the field by offering researchers valuable insights into the spatial context of complex cellular processes and interactions. By analyzing multiple targets in the same specimen, researchers can obtain a holistic view of their tissue while conserving precious sample. However, performing multiplex assays can be challenging. Cross reactivity between detection reagents can often limit reagent choices and circumventing this by directly labeling primary antibodies can lead to altered antibody function and loss of specificity. Thankfully, VectaPlex™ Antibody Removal Kit comes to the rescue, simplifying the multiplex workflow, preserving tissue integrity, and enabling multiple rounds of staining without reducing target availability. 

VectaPlex Antibody Removal Kit is a versatile solution designed to remove antibodies and other non-covalently bound reagents from formalin-fixed paraffin-embedded (FFPE) tissue sections after staining in both immunofluorescence (IF) and tyramide signal amplification workflows. The kit’s gentle yet efficient removal process preserves tissue integrity and morphology through multiple cycles of antibody binding and removal, ensuring that it maintains the quality of the samples throughout the entire process. Read below on how exactly to use the VectaPlex kit. 

Step-by-step directions for using VectaPlex 

Option 1: Immunofluorescence Workflow 

1. Complete the standard immunofluorescence staining procedure, mount the slide with a non-hardening (non-setting) mounting medium, and image. 
2. Remove the coverslip from the tissue section and wash thoroughly with PBS to remove any residual mounting medium. 
3. Add VectaPlex Reagent A to cover the tissue section completely (typically ~ 150–200 μL) and incubate for 15 minutes at room temperature. 
4. Wash the slides briefly with PBS. 
5. Add VectaPlex Reagent B to cover the tissue section completely (typically ~ 150–200 μL) and incubate for 15 minutes at room temperature. 
6. Wash the slides with PBS for 5 minutes. 
7. The tissue section is now ready to begin another immunofluorescence staining procedure with a protein blocking step followed by the next round of antibody staining, mounting, and imaging. 
8. Continue repeating these steps until you have detected all desired antigens. 

Note: We recommend using a mounting medium containing DAPI during each staining cycle for the IF workflow to facilitate image alignment.  

Note: You can register and overlay images from successive staining cycles using free software such as Fiji. 

Option 2: Tyramide Signal Amplification Workflow 

1. Complete the standard tyramide signal amplification staining procedure. 
2. Add VectaPlex Reagent A to cover the tissue section completely (typically ~ 150–200 μL) and incubate for 15 minutes at room temperature. 
3. Wash the slides briefly with PBS. 
4. Add VectaPlex Reagent B to cover the tissue section completely (typically ~ 150–200 μL) and incubate for 15 minutes at room temperature. 
5. Wash the slides with PBS for 5 minutes. 
6. The tissue section is now ready to begin another fluorescent tyramide signal amplification staining procedure using a differently colored dye. 
7. Repeat these steps until you have detected all desired antigens. 
8. After completing the staining process, mount the tissue sections and capture images. 

It is important to note when using the VectaPlex reagents that mixing with bleach may result in formation of a hazardous gas. For this reason, do not mix reagents or waste solutions from this process with bleach, and use reagents in a well-ventilated area using appropriate laboratory personal protective equipment (PPE). 

Benefits of VectaPlex Antibody Removal Kit 

The VectaPlex Antibody Removal Kit offers a range of benefits that make it an indispensable tool for multiplex workflows, such as: 

• Effective Antibody Removal: VectaPlex efficiently removes antibodies and non-covalently bound detection reagents, minimizing carryover between staining rounds and ensuring accurate results. 
• Preservation of Tissue Integrity: The gentle removal process maintains tissue morphology and integrity, enabling multiple rounds of staining without compromising sample quality. 
• Enhanced Multiplexing: VectaPlex enables up to six rounds of staining without reducing target availability, providing researchers with deeper insights into complex biological processes. 
• Ready-to-Use Convenience: VectaPlex comes ready-to-use, streamlining the workflow and saving time in the laboratory. 

We hope that this blog post has provided you with valuable insights into the VectaPlex Antibody Removal Kit and its significance in simplifying multiplex workflows. By following the user-friendly steps outlined above, you can effectively remove antibodies and non-covalently bound detection reagents, allowing you to achieve your multiplexing goals with ease.  

For more resources and tips on enhancing your multiplexing endeavors, check out our IHC Multiplexing Resource Guide, the IF Guide, and other tips and tricks available on our blog. 

Have you ever attempted to get immunofluorescent image of a tissue section only to be met with a black image on the microscope? Your tissue section might be the victim of photobleaching.  

What is photobleaching? Photobleaching happens when a fluorophore permanently loses its ability to emit a fluorescent signal and occurs as a byproduct of the mechanisms underlying fluorescence emission (1,2). Each fluorophore can undergo a different number of excitation and emission cycles until they permanently stay in an excited state and lose the ability to emit fluorescence (1,2) As photobleaching happens, it becomes increasingly difficult to image an IF experiment. This progressive reduction in signal intensity also increases the possibility of false-negative results and reduces the feasibility of experiments targeting quantitative analyses.  

Taking precautionary measures when mounting and visualizing immunofluorescence (IF) experiments is essential to protect the fluorescent signal from photobleaching. This blog post will walk you through strategies in four different categories to protect your immunofluorescent tissue section from photobleaching. Keep reading for tips, and if you need help with other steps in the IF mounting process, be sure to check out our Immunofluorescence Mounting Guide. 

Light Intensity 

The higher the intensity of the light used during specimen visualization, the quicker electrons go through multiple cycles of excitation and emission, directly contributing to photobleaching. Light intensity refers to the power of the light source, also known as its brightness. Thus, one of the most straightforward strategies to reduce photobleaching and extend the life of the fluorophore is to reduce the intensity of the light during imaging (3). 

An easy solution to reducing photobleaching in mercury and xenon-arc lamps is to use neutral-density filters. These accessories reduce light intensity by a given percentage that varies from filter to filter. For example, a neutral-density filter with 80% transmittance allows only 80% of the light to reach the sample. 

Alternatively, fluorescence microscopes can use manually adjustable LED lamps as the light source. With either the addition of filters or manual light intensity reduction, fewer protons reach the sample, resulting in less excitation and a dimmer image. Keep in mind, reducing the light intensity might not be worth it if longer exposure is required to acquire an image with an appropriate fluorescent signal. 

Image Exposure Time 

Exposure is the time the camera shutter needs to remain open to properly image the specimen. Longer exposure yields brighter images but also increases the amount of light reaching the sample and accelerates photobleaching. Shorter exposures can result in dim images.  

When reduced exposure results in a dim, unsuitable image, increasing the gain function of the photomultiplier can help increase the overall signal intensity. Gain measures the amplification of the signal reaching the camera sensor. Higher gain yields a brighter image while keeping the light intensity and exposure constant. This approach increases camera sensitivity to light in a non-selective way, which means that background noise will also become brighter. In addition, higher gain can also contribute to reduced image resolution. Therefore, the perfect balance between light intensity, exposure time, and gain should be determined using a test section while keeping tissue type, target protein, and fluorescent dye constant. 

Using an objective with a wider aperture is one final approach to increase the amount of light reaching the sample. Objectives with a high numerical aperture allow more light to reach the sample, and as a result, the researcher can reduce light intensity, exposure, and gain and still acquire an image with adequate signal intensity. Keep in mind, wider apertures can also result in a smaller depth of field, which can be beneficial or detrimental, depending on the objectives of the experiment. 

Fluorophore Selection 

Different fluorophores have varied susceptibility to photobleaching. Newer generations of fluorophores, such as AlexaFluor® and DyLight, have more stable structures and are less susceptible to photobleaching than traditional fluorophores such as FITC, TRITC, and Texas Red™ (4). Changing the type of fluorophore is a straightforward strategy to reduce photobleaching during image visualization. 

When performing multi-color staining, the researcher should select fluorophores with minimal spectral overlap, otherwise a single channel can excite multiple fluorophores with overlapping excitation spectra, leading to bleed-through and photobleaching (1,2). Manufacturers often report the excitation and emission spectra for each fluorophore, and this information should be carefully considered when designing an experiment. Choosing fluorophores with narrow excitation and emission spectra can also help reduce overlap. Many free online tools are available to help with fluorophore selection for multi-color staining.  

Fluorophores also vary in their sensitivity to light—they require different intensities of light to yield a fluorescent signal with similar strength. Thus, when considering all the factors influencing the choice of fluorophores, the best option combines good sensitivity and less photobleaching. 

Antifade Mounting Media 

Fading is another word commonly used to describe photobleaching. Thus, mounting media that confer protection against photobleaching are known as antifade mounting media. Adding these reagents during the mounting process is the most effective way to prevent photobleaching.  

Antifade mounting media prevent the reaction of excited electrons with other molecules and thus extend the number of excitation/emission cycles they can undergo (5). In addition, mounting media maximize fluorophore signal strength, requiring lower light intensity and exposure for optimal specimen visualization (5).  

Other benefits of mounting media include preservation of tissue morphology and improved image quality (6). As many different options for mounting media are available, researchers should be aware of the many factors influencing the final choice and their impacts on the final results. To learn more about selecting the right antifade mounting media, check out our blog post How to choose antifade mounting media.  

We hope the above tips were helpful and get you on the road to better immunofluorescence images. For more tips and tricks to improve your IF staining, check out our IF Resources Guide and watch the video below on how to coverslip your IF slides to protect your fluorescence.  

References 

1. Vicente NB, et al. 2007. Photobleaching Correction in Fluorescence Microscopy Images. Journal of Physics: Conference Series.
2. Demchenko AP. 2020. Photobleaching of Organic Fluorophores: Quantitative Characterization, Mechanisms, Protection. Methods and Applications in Fluorescence.
3. Hoebe RA, et al. 2007. Controlled Light-Exposure Microscopy Reduces Photobleaching and Phototoxicity in Fluorescence Live-Cell Imaging. Nature Biotechnology.
4. Zheng Q, et al. 2014. Ultra-Stable Organic Fluorophores for Single-Molecule Research. Chemical Society Reviews.
5. Collins TJ. 2006. Mounting Media and Antifade Reagents. Microscopy Today.
6. Ravikumar S, et al. 2014. Mounting Media: An Overview. Journal of Dr. NTR. University of Health Sciences.

Watch the below video for tips on coverslipping your immunofluorescent slides.