Webinar | Simplifying Multiplex IF with Your Validated Primary Antibodies and AI-Powered Spatial Analysis
Lab Helper

Your bench companion.

New

AI Image Analysis

From stained slide to quantified data, without leaving your workflow.

Discover more
Reagent calculator

Calculate your Connector & antibody volumes

Pick a Revolune Connector and primary antibody for each biomarker. Then, enter the stock concentration and working concentration of your primary antibody as well as your antibody cocktail working solution. The tool calculates the volume of primary antibody and Revolune Connector needed.

i
The calculation works the same for both anti-rabbit and anti-mouse Revolune Connectors.

Resources

Want the technical details?

Download our manual and automation-ready protocols to see how easily Illuminate Reagents can fit into your multiplex IF workflow.

Manual staining protocols

Illuminate P4-R - Manual staining protocol
Download
Illuminate P5-R - Manual staining protocol
Download
Illuminate P6-R - Manual staining protocol
Download
Illuminate P7-R - Manual staining protocol
Download

Automated staining — Bond RX

Illuminate P4-R - Bond RX protocol
Download
Illuminate P5-R - Bond RX protocol
Download
Illuminate P6-R - Bond RX protocol
Download
Illuminate P7-R - Bond RX protocol
Download

Reagents Calculator

Calculate now

FAQs

Choosing the right multiplex immunofluorescence workflow often raises important questions around compatibility, assay development, imaging requirements, and scalability. Explore the answers to the most common questions about Illuminate Reagents and discover how our technology fits into existing spatial biology workflows.

Thank you! Your submission has been received!
Oops! Something went wrong while submitting the form.
Is this clinically validated?

Currently for research use only. However, the workflow is designed to be compatible with clinical lab requirements such as automation and reproducibility.

Multiplexing
Why not use TSA since it is well established?

TSA is effective but involves complex, time-consuming workflows and can damage tissue through repeated cycles. This approach provides comparable sensitivity with a simpler, faster, and less harsh workflow.

Multiplexing
What level of multiplexing is supported?

A 4-plex kit is available now, with 5- to 7-plex kits coming soon. The DNA-based system is inherently scalable.

Workflow Integration
Is there special mounting medium required?

Yes, ProLong Gold mounting medium

Workflow Integration
For the 4-plex, what wavelength is required for imaging?

You need wavelengths of 488,555,647 and 750

How long does a run take?

A typical 4-plex assay can be completed in approximately 6 hours, significantly faster than traditional multiplex approaches.

Workflow Integration
Are new instruments required?

No.The system is compatible with standard fluorescence microscopes and existing lab infrastructure. No proprietary hardware is needed.

Workflow Integration
How reproducible is the assay?

Highly reproducible.Parallel staining reduces variability, and the workflow is compatible with automation. Fewer steps result in fewer potential sources of error. Furthermore, the stainng protocol is automatable and can be run on an autostainer to increase reproducibility.

Tissue Integrity and Reproducibility
How does the technology work?

Primary antibodies are pre-incubated with Revolune Connectors which act as anchors for signal amplification. A DNA scaffold is then built at each antibody site, allowing multiple fluorescent probes to bind. This results in strong signal amplification while enabling all antibodies to be applied simultaneously in one step.

Workflow and Technology
What differentiates this from TSA or cyclic IF?

TSA relies on sequential staining and enzymatic amplification, which increases time and complexity. Cyclic IF requires repeated stripping, which can damage tissue. Revolune enables parallel staining with DNA-based amplification, eliminating stripping and reducing workflow time significantly.

Workflow and Technology
Is this truly a single-step staining process?

Antibodies are first pre-incubated with Revolune Connectors. After that, all antibodies are applied simultaneously, followed by amplification and detection. This removes the need for iterative staining cycles.

Workflow and Technology
Do antibodies need to be conjugated?

No. The system works with unmodified, pre-validated primary antibodies. Revolune Connector binding is achieved through a simple pre-incubation step, preserving existing validation and workflows.

Antibodies and Flexibility
Can any antibody be used?

Yes, within species compatibility.Current kits support rabbit antibodies, with expansion to mouse antibodies soon. No custom conjugation or proprietary antibody panels are required.

Antibodies and Flexibility
Do panels need to be redesigned?

No.Existing antibody panels can be reused. There are no strict species pairing constraints, allowing flexible panel design and faster assay development.

Antibodies and Flexibility
How is strong signal achieved without TSA?

DNA scaffold-based amplification. 

Each antibody site builds a DNA scaffold that can bind multiple fluorescent probes. This increases signal intensity and improves signal-to-noise ratio.

Signal and Data Quality
What about background and specificity?

Signal is generated through DNA-based amplification rather than diffusion-based deposition. This results in low background and high specificity, leading to cleaner staining and improved spatial accuracy.

Signal and Data Quality
Does amplification affect spatial resolution?

Amplification is localized at the antibody binding site. The DNA scaffold remains spatially constrained, preserving subcellular resolution.

Signal and Data Quality
Does the workflow damage tissue?

No.
There is no antibody stripping or harsh treatment involved. This preserves tissue morphology and antigen integrity, which is especially important for sensitive samples.

Tissue Integrity and Reproducibility

Still have questions?

Get in touch with our team to discuss your application, research goals, or technical requirements. We're happy to help you find the right solution for your workflow.