Multiplex immunohistochemistry (IHC) allows for the simultaneous detection of multiple tissue biomarkers for proteomics profiling. One of the popular technologies is Opal Multiplex IHC for proteomics profiling. However, the overall manual staining process is very lengthy and laborious. Opal multiplex IHC workflow combines standard unlabeled primary antibody with Tyramide Signal Amplification (TSA) and the Opal polymer horseradish peroxidase (HRP) to amplify the IHC signal and enables the visualization of biomarkers by covalently depositing fluorescent molecules close to the antigen of interest (Figure 1). Proteomics profiling using same tissue section provides in-depth details of molecular signaling and its surrounding microenvironment.
In this blog, we highlight recent milestones we have achieved to automate the Opal multiplex IHC protocol on NanoVIP®, our state-of-the-art fully automated staining system, where we significantly reduced processing time, hands-on time, and antibody volume.
Manual Opal Multiplexing IHC Staining Protocol
Assay Development (pre-staining): Before running Opal multiplexing IHC protocols, users must perform control slides testing to determine denaturing time or temperature for antigen retrieval. This is part of the assay development process for antibody optimization. This process includes antigen retrieval buffers parameters, determining correct antibody titrations, antibody-Opal-fluorophore pairings, and antibody stripping steps incorporated into your protocol.
Staining Process: After optimization of marker concentrations and panel design, the Opal 6-plex + DAPI staining protocol is estimated to require over 14 hours of hands-on time, from preliminary steps to applying final coverslip. This does not include time spent developing antibody optimization. Because this protocol involves pipetting by hand, we estimate that users will need about 200 μl of antibody volume.
Automation with NanoVIP®
The automated staining process on NanoVIP® streamlines the manual Opal protocol and reduces hands-on time and reagent volumes (Figure 2). However, users must still perform the assay development steps to determine correct titrations and antibody-Opal fluorophore pairings. One advantage of NanoVIP® is that antigen retrieval is also done within the instrument. Therefore, there is no need for an additional instrument or determining antigen retrieval buffer parameters.
- All steps are performed by NanoVIP®—Hands-on steps involve mixing the appropriate reagents and solutions for loading.
- Estimated hands-on time for 6-plex + DAPI: < 1 hour—This time is an estimation of collecting and filling reagent vials and buffers.
- Once users load reagents, buffers, and slides into NanoVIP®, there is no more hands-on time until the final removal of slide for imaging.
- 25 μl of antibody volume required for NanoVIP® instead of 150-200 μl of manual staining requirements.
The automation with NanoVIP® for manual staining process has several advantages, including:
- Cost-effectiveness: Reducing reagent costs and labor required compared to conventional methods.
- Time-efficiency: Automating manual staining steps saves considerable time and generates consistent results with antigen retrieval done within the instrument; and
- Streamlined Process: Using automated processes simplifies the protocol, making it more accessible to researchers.
The Opal staining system allows for multiplexing six biomarkers in the same tissue sample simultaneously. However, manual protocols require more hands-on time and higher reagent volumes. Omicsveu has automated Opal manual protocol using NanoVIP® and reduced processing time from > 48 to < 12 hours, hands-on time from > 14 to < 1 hour, and antibody volumes from 200 μl to 25 μl while ensuring consistency with clean, crisp, and intense staining results.
A comparison between Opal’s manual protocol and the automation of NanoVIP® is illustrated below.
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