How to use VectaPlex™ Antibody Removal Kit

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.