Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

This protocol is significant because the development optimization and transfer of multiplex immunofluorescence protocols is complex and challenging. For many labs the toe hold of an optimized protocol is invaluable. Multi-spectral immunofluorescence allows us to assess both the location and the identity of multiple cell types in and around the tumor.

This is a powerful combination of information in immune oncology studies. The most compelling use for multiplex immunofluorescence is immune oncology. But the ability to survey multiple immune cell types in setu can be leveraged across all auto-immune and inflammatory therapies.

Knowing the location and phenotype of immuno cells in and around solidual cells is both prognostic and predictable responses to tumor specific immunotherapy. The optimization and validation of the protocol are key steps that should not be rushed. New developers should familiarize themselves with the new kinds of artifacts associated with multi-spectral imaging.

To locate the base multi-plex protocol preload it on the automated stainer. Filter for all'instead of preferred'on the protocol screen. Before making changes save the original protocol and click the protocols tab, and right click Opal 6 Multiplex to select copy.

Change the name to 7 Multiplex Protocol 1'in the new window and select the tab associated with the correct device. Match the protocol in supplemental table S1'and click add reagent to add a ten minute paroxidies inhibition step, selecting the peroxidase inhibitor as the reagent. Confirm that the blocking steps utilize a protein kinase inhibitor blocking buffer, that each primary anti-body refers to the appropriate reagent, that the secondary anti-body steps utilize a horseradish peroxidase polymer and that the spectral dapi provided with the multi-spectral automation kit is utilized.

Make sure that the protocol contains the peroxidase inhibitor, followed by six sequential staining runs each containing a protein blocking step, a primary anti-body, a secondary anti-body, a Tyramide floor, and then antigen retrieval to remove the anti-bodies. To add the drop controls, copy the 7 Multiplex Protocol 1'name, and change it to 7 Multiplex Protocol 1 CD68 Drop. Change the CD68 anti-body reagent to Mouse IgG and save the protocol.

Then, create six more new protocols, one for each drop control, and one for the complete iso-type control in the same manner. Make a drop control protocol for each target in the same way as I've shown you here. To prepare a research detection kit, fill the 30 milliliter open container marked for 1X tris-buffered saline with 1X tris-buffered saline, and place the container in the position farthest from the handle in the Multiplex Research Kit 1.

Label two 30 milliliter open containers muli-spectral block'and multi-spectral secondary, and fill the mutli-spectral block container with 30 milliliters of blocking buffer antibody diluent from the Multi-Spectral Staining Kit. Fill the multi-spectral secondary container with 30 milliliters of anti-Mouse anti-rabid secondary antibody polymer from the Multi-spectral Staining Kit and add 600 microliters of antibody diluent to each of ten 7 milliliter open containers. Next, add the concentrated primary antibody to the appropriate tubes in the volumes indicated in the table, and mix by gentle pipetting.

Add 3 milliliters of double distilled water, plus 12 drops of spectral dapi into one 7 milliliter open container, and 3 milliliters of peroxidase block into a second 7 milliliter open container. Re-suspend each liathalized floor with 75 microliters of dimethyl sulfoxide and mix by pipetting. Then, store the Tyramide signal amplification floor stocks at 4 degrees Celsius until ready.

While preparing the individual TSA floor dilutions. Register all reagents on the auto-stainer, then place each container into a reagent rack and load all of the reagents onto the automated stainer. The stainer will detect the presence and confirm the volume of each reagent.

Once all of the reagents have been loaded, activate the protocol to run overnight. At the end of the staining procedure, mount the samples with cover slips and load the samples into the Multi-Spectral Imaging Platform Scanner for scanning. When all of the samples have been scanned, the images will be formatted as qptiff files.

Open an appropriate qptiff analysis software program, and click load slide'to open a five filter whole slide scan of the slide of interest. Log in and use the stamp tool to select five regions for multi-spectral imaging. Under Select for:select Acquisition'under Size in Fields:select 1x1 and under Field resolution'select 0.5 micrometers 20x.

Based on this cytokeratin staining select several regions with both cytokeratin positive tumor tissue, and cytokeratin negative stromal tissue to be imaged from the overall scan. When all of the multi spectral images have been selected, switch to the Vectra software and change the task from scan'to multi-spectral imaging'for each slide. Then, click scan to perform the multi-spectral imaging collection.

When the multi-spectral image acquisition is complete, use the spectral un-mixing software to open files within the multi-spectral image folder. Under image format'select multi-spectral'Under sample format'select fluorescence'And under Spectral Library Source'select inForm'To select the floors, select and load all six floors, and dapi from the sample library slides, and click prepare all. The spectral un-mixing software will perform spectral unmixing on all of the open images using the provided spectral profiles.

Identify and annotate the auto-fluorescent spectral profile. Be sure to check the auto-fluorescent feature to insure that it is completely captured in the auto-fluorescent channel after un-mixing and to test different samplings until it is removed acceptably. Then assess the staining pattern against the chromogenic single stains of the same target in sequential slides of the same tissue with a pathologist.

To assess the fluorescence intensity of each channel, use the counts tool to survey the open images. Then, return to the staining protocol, and adjust the TSA concentration to address any normalized counts that exceed or fail to reach the acceptable range. In this representative analysis the tonsil tissue demonstrated a clearly defined cytokeratin staining within the tonsil surface, squamous epithelium without background in the lymphoid tissue with cytokeratin and PDL-1 staining apparent in the reticulated epithelium of the crypts.

The follicular germinal centers were easily recognizable and rich in Ki-67 positive lymphocytes. The macrophages present within the germinal centers were identified by their CD-68 expression with some macrophages also observed outside of the germinal center. CD-8 stained lymphocytes with a T-cell distribution and PD-1 expression strongly stained small lymphocytes clustered at the periphery of the germinal center, as well as scattered throughout the interfollicular region.

After the expected staining pattern was confirmed in the tonsil tissue control, the same staining could then be evaluated in a lung cancer sample. Isotype negative controls and drop controls should also be evaluated;not only for the detection of background and auto-fluorescence, but also for umbrella effects and spectral bleed. Drop controls are important for evaluating potential artifacts in the staining due to the multi-plex immunofluorescence method during the optimization of a new multi-plex panel.

Each drop control should show the same staining pattern as the full multi-plex panel, except for the single primary antibody which should be removed on each specific control. Immuno profiling with multi-spectral immunofluorescence allows for the stratification of tumors and regions by immune criteria which can then be investigated by massively multiplex genomic, transcriptomic, and proteomic studies. I've used multi-plex immunofluorescence to investigate mechanisms of action in oncology and auto-immune disorders which allows for the identification and characterization of immune and pathogenic cells in a single section in setu.

General precautions should be taken when working with anti-bodies against human proteins, Tyramide and dapi, but no reagent or material in this protocol is known to be particularly hazardous.