High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research

We're developing kits for rapid and sensitive multiplex immunofluorescence assays. The goal here is to help researchers profile different cell types appearing in FFPE tissue

Immunofluorescence is commonly carried out using dye-labeled or peroxidase-labeled secondary antibodies. Immunohistochemistry tends to be low plex. Dye-labeled secondary antibodies suffer from lower signal. Tyramide signal amplification assays are laborious to optimize and take several hours to multiple days to run.

The assays' markers come pre-optimized and it is significantly faster than other techniques. Standing four markers takes less than eight hours, and about three more hours for every additional four biomarkers.

The ability to run multiplex immunofluorescence faster will enable spatial proteomics to be more widely applied and will reduce the barrier to understanding the spatial contexture of tissue.

[Narrator] To begin, thaw all the kit reagents at room temperature. Store the antibody conjugates, amplification enzyme, exchange initiator, and exchange neutralizer at minus 20 degrees Celsius until use. Vortex a thawed antibody diluent. Pipette the antibody diluent into a container. Briefly centrifuge and add all antibodies into the antibody staining solution 1 container. Add in the provided antibody diluent at a 1 to 100 dilution. Then use a pipette to mix the solution gently, avoiding vortexing. Vortex the pre-amplification mixed solution after thawing to mix thoroughly. Then add the solution into the respective mix container. Vortex the amplification buffer and pipette mix the amplification enzyme. Dilute the amplification enzyme at a 1 to 10 ratio in the amplification buffer to create the amplification buffer. Mix gently by pipetting, avoiding vortexing. Then vortex and centrifuge the provided nuclear counterstain solution and dilute 1 to 100 in ultrapure water. Next, dilute fluorescent probes 1 solution in a 1 to 20 ratio in probe buffer. Insert the full reagent wand into the instrument to allow reagent volumes to be measured. Navigate to the Slide setup tab at the top of the auto stainer software once all reagents are prepared. Click the Add study button and input the study ID, study name, and any study comments as needed. Under Dispense volume, select 150 microliters and choose *Dewax 4 Steps as the preparation method. If it is not available in the dropdown, open the Protocol setup menu, mark the preferred checkbox for it, and click OK. After the study is created, click on the Add slide button located on the right side of the Slide setup screen. Enter a unique name for the slide under Slide comments. Set the Tissue type to Test tissue in the appropriate field. Then choose 150 microliters for Dispense volume and set the Staining mode to Single in the left dropdown and Routine in the right dropdown. Under Process, choose IHC, and set the Marker to antibody staining solution. In the Protocols section of the Add slide window, select Staining Assay 1 for Staining, *Dewax 4 Steps for Preparation, and *HIER 20 minutes with ER2 For HIER. Leave the Enzyme protocol set to asterisk dash, and click Add slide when all fields are complete. After adding all slides, close the Add slide window and print slide labels, affixing each to the corresponding tissue section. Load each labeled slide onto the tray and place cover tiles on top of each tissue section. Insert the slide tray into the instrument and select the corresponding auto stainer from the right side of the software screen. Confirm that all three slide trays and all reagent information, including bulk and prepared reagents, are visible. Check that the bulk reagent containers are adequately filled and that waste containers have enough volume to complete the run. Then press the play button to begin the staining run and ensure timely removal of the slides from the auto stainer once the procedure is complete. Place stained slides in PBS buffer and apply cover slips using the mounting medium. Image the slides using a fluorescence microscope or whole slide scanner with filters compatible with the assay dyes. For the exchange assay, thaw the exchange buffer at room temperature. Keep the exchange neutralizer and exchange initiator stored minus 20 degrees Celsius until ready for use. Prepare two reagent titration containers and retrieve the detection kit for use during the exchange protocol. Vortex the exchange buffer. And pipette mix the exchange initiator. Prepare the exchange solution in its designated container by mixing required volumes of exchange initiator and exchange buffer. Mix the contents by pipetting only. Now combine the fluorescent probes 2 solution with the diluted exchange buffer in the respective container. Vortex only the probe buffer and fluorescent probe mix and pipette mix the final solution. Place the full reagent wand with all prepared reagents onto the auto stainer. Allow the machine to perform a dip test on all reagents. After adding the slides, set the marker to *Negative. And for Preparation, HIER, and Enzyme, select the asterisk dash option, since no dewaxing or antigen retrieval is needed for the exchange protocol. Click Add slide when complete. After all slides are added, close the Add slide window and print slide labels to be affixed to each corresponding tissue section. Load each slide onto the tray and place a cover tile over each tissue section before placing the tray into the auto stainer. Now press the play button to initiate the run. Images of a tissue microarray core captured across two staining rounds using an eight-plex immunofluorescence protocol showed distinct immune markers in each round, with successful image co-registration combining both rounds into a unified composite. Whole slide imaging of colorectal cancer tissue enabled identification of immune phenotypes based on colocalization of markers, including regulatory T cells, exhausted T cells, and immune suppressive macrophages. Immunofluorescence staining showed co-expression of CD3, CD4, and FoxP3 in regulatory T cells. PD1 and PDL1 in tumor and stromal cells. CD3, CD8, and PD1 in exhausted cytotoxic T cells. And CD 68 and PDL1 in immune-suppressive macrophages. Comparison of single marker immunohistochemistry and single or multiplex immunofluorescence confirmed qualitative concordance in staining patterns across all markers. Serial section staining across different tissue types confirmed consistent expression patterns of immune markers with representative tissue cores, showing successful staining and minimal variability. Tonsil sections stained in six serial slides for CD8 demonstrated high reproducibility with nearly identical signal distribution across all images. Quantification of marker positive cell densities across tissue types showed highest densities in tonsil and lymph node, and lowest in melanoma and colon.