This protocol helps to guide the selection of regions of interest for spatial omics technologies, which allows a more targeted characterization of tissues or cell populations. An automated protocol for ROI selection in proteomics assays is more robust and reproducible than manual protocols, and using 50 micrometer ROIs for transcriptomics assays allows profiling of specific cell populations. Start with programming the autostainer to apply fluorescent visualization antibodies.
In the autostainer software, click on the home button and choose Protocols. Then click on Create/Edit Protocols and select RUO Discovery Universal as the procedure. Click on First Sequence, then select Deparaffinazation, followed by Depar v2.
For Medium Temperature, choose 72 degrees Celsius, then click on Pretreatment and select Cell Conditioning and CC1 Reservoir. For Very High Temperature, choose 100 degrees Celsius, then click on CC1 eight minutes, and continue clicking until CC1 64 minutes is selected. Click on Inhibitor, then select DISCOVERY Inhibitor, and for Incubation Time, choose eight minutes.
Then click on Antibody, followed by High-Temp Antibody Incubation. For Low Temperature, choose 37 degrees Celsius. For Antibody, choose the ANTIBODY 6 from the dispenser label.
For Plus Incubation Time, select 32 minutes. Click on Multimer HRP, then select Multimer HRP Blocker. Then for Antibody Blocking, choose Gt Ig Block.
And for Incubation Time, choose four minutes. Next, on Multimer HRP Reagent, select OMap Anti-Rb HRP, and for Incubation Time, choose 16 minutes. Then click on Cy5, and for Long Incubation Time, choose zero hour, eight minutes.
Click on Dual Sequence and choose Antibody Denaturation. Then select Antibody Denature CC2-1, and for a Very High Temperature, choose 100 degrees Celsius. Ensure that incubation time is eight minutes.
Next, click on DS Inhibitor and select Neutralize. Click on DS Antibody, and for Very Low Temperature, choose 37 degrees Celsius. Then in Antibody, choose ANTIBODY 3 from the dispenser label.
And for Plus Incubation Time, select 32 minutes. Click on DS Multimer HRP and choose DS Multimer HRP Blocker. Then for Antibody Blocking, select Gt Ig Block, and for Incubation Time, choose four minutes.
Next, on Multimer HRP Reagent, select OMap anti-Ms HRP, and for Incubation Time, choose 16 minutes. Then click on DS Rhodamine 6G, and for Long Incubation Time, choose zero hour, eight minutes. Click on Triple Stain and select TS Antibody Denaturation, then select Antibody Denature CC2-2, and for Very High Temperature, select 100 degrees Celsius.
Ensure that incubation time is eight minutes. Next, click on TS Inhibitor and select TS Neutralize. Click on TS Antibody, and for Very Low Temperature, select 37 degrees Celsius.
Then in Antibody, select the ANTIBODY 7 from the dispenser label, and for Plus Incubation Time, select 32 minutes. Click on TS Multimer HRP and choose TS Multimer HRP Blocker. Then for Antibody Blocking, select Gt Ig Block, and for Incubation Time, choose four minutes.
Next on Multimer HRP Reagent, select OMap Anti-Rb HRP, and for Incubation Time, choose 16 minutes. Then click on TS FAM, and for Long Incubation Time, choose zero hour, eight minutes. Click on Save and add a title to the protocol.
Select a protocol number, add a comment, and click on Active, followed by Save. Bake vendor-procured formalin-fixed paraffin-embedded human tissue sections in an oven set to 70 degrees Celsius for 20 to 60 minutes. While slides are baking, print labels by clicking Create Label in the autostainer software.
Next, click on Protocols, select the protocol number and click on Close/Print. Add relevant information on the slide label and click on Print. Remove slides from the oven and let them cool down to room temperature.
Apply the previously printed protocol labels to the corresponding slides. Load refillable antibody dispensers with antibodies according to the antibody label numbers. Dilute each antibody in the specified diluent and prime the refillable antibody dispensers.
Gather blocking, detection, and amplification prefilled reagent dispensers and place them on the instrument reagent tray. Load slides in the slide drawers and click on Running, followed by Yes. Confirm the start of the run by checking the run duration on the autostainer software.
The next day, ensure run completion by observing green flashing lights on the slide drawer slots. Then take the slides off the instrument and rinse the slides vigorously in 1x reaction buffer until liquid cover slip solution is completely removed. Replace SYTO 13 with SYTO 64 at 5, 000 nanomolar to enable fluorophore integration.
Dilute SYTO 64 in 1x TBS and incubate in a humidity chamber for 15 minutes. Use FITC for DISCOVERY Fam and set the exposure to 200 milliseconds. Use Cy3 for DISCOVERY Rhodamine 6G and set the exposure to 200 milliseconds.
Use Texas Red for SYTO 64 and set the exposure to 50 milliseconds. Specify SYTO 64 as the focus channel, and finally, use Cy5 for DISCOVERY Cy5. Set the exposure to 200 milliseconds and save changes.
Bake formalin-fixed paraffin-embedded cell pellet sections in an oven set to 70 degrees Celsius for 20 to 60 minutes. Scan slides on the spatial profiling platform and select sizes of 50 micrometers and 300 micrometers. The automated visualization protocol was developed by including leave one out controls to confirm that there was no bleed through from the neighboring channels.
The log 2 heat map of all the markers included in the spatial proteomics assay comparing the manual pan-cytokeratin CD 45 protocol in black to the automated 3-plex protocol in gray shows a Spearman R value of 0.88. Of the 31 antibodies used in the assays, 23 antibodies had a Spearman R value higher than or equal to 0.5. The log 2 heat map of selected targets included in the spatial transcriptomics assay comparing the manual pan-cytokeratin CD 45 protocol in black to the 3-plex automated protocol in gray showed differences in these values.
The sequencing data for the 3-plex automated visualization protocol presented lower values when compared to the pan-cytokeratin CD 45 manual visualization protocol, which is also reflected by the low Spearman R values of 0.15. Also, a loss of dynamic range was observed when using the 3-plex visualization automated protocol. The detection of small ROIs in the spatial transcriptomics protocol was performed by the selection of circular regions of interest at 50 micrometer and 300 micrometer diameter RNA counts for selected targets on different cell lines were comparable between the two regions of interest sizes after normalization.
This protocol for automated visualization panels can be adapted by researchers by exchanging markers in order to develop panels that precisely define ROIs to answer their spatial proteomics questions.
